Microorganisms and processes for enhanced production of pantothenate

ABSTRACT

The present invention features improved methods for the enhanced production of pantoate and pantothenate utilizing microorganisms having modified pantothenate biosynthetic enzyme activities and having modified methylenetetrahydrofolate (MTF) biosynthetic enzyme activities. In particular, the invention features methods for enhancing production of desired products by increasing levels of a key intermediate, ketopantoate, by increasing enzymes or substrates that contribute directly or indirectly to its synthesis. Recombinant microorganisms and conditions for culturing same are also are featured. Also featured are compositions produced by such microorganisms.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/393,826, filed on Jul. 3, 2002, entitled “Microorganisms andProcesses for Enhanced Production of Pantothenate”. This application isrelated to International Patent Application No. PCT/US02/00925, entitled“Microorganisms and Processes for Enhanced Production of Pantothenate”,filed Jan. 18, 2002 (pending), which, in turn, claims the benefit ofprior-filed provisional Patent Application Ser. No. 60/347,638, entitled“Microorganisms and Processes for Enhanced Production of Pantothenate”,filed Jan. 11, 2002, to prior-filed provisional Patent Application Ser.No. 60/263,053, filed Jan. 19, 2001 (expired), and to prior-filedprovisional Patent Application Ser. No. 60/262,995, filed Jan. 19, 2001(expired). The present invention is also related to U.S. patentapplication Ser. No. 09/667,569, filed Sep. 21, 2000 (pending), which isa continuation-in-part of U.S. patent application Ser. No. 09/400,494,filed Sep. 21, 1999 (abandoned). U.S. patent application Ser. No.09/667,569 also claims the benefit of prior-filed provisional PatentApplication Ser. No. 60/210,072, filed Jun. 7, 2000 (expired),provisional Patent Application Ser. No. 60/221,836, filed Jul. 28, 2000(expired), and provisional Patent Application Ser. No. 60/227,860, filedAug. 24, 2000 (expired). The entire content of each of theabove-referenced applications is incorporated herein by this reference.

BACKGROUND OF THE INVENTION

Pantothenate also known as pantothenic acid or vitamin B5, is a memberof the B complex of vitamins and is a nutritional requirement formammals, including livestock and humans (e.g., from food sources, as awater soluble vitamin supplement or as a feed additive). In cells,pantothenate is used primarily for the biosynthesis of coenzyme A (CoA)and acyl carrier protein (ACP). These coenzymes function in themetabolism of acyl moieties which form thioesters with the sulfhydrylgroup of the 4′-phosphopantetheine portion of these molecules. Thesecoenzymes are essential in all cells, participating in over 100different intermediary reactions in cellular metabolism.

The conventional means of synthesizing pantothenate (in particular, thebioactive D isomer) is via chemical synthesis from bulk chemicals, aprocess which is hampered by excessive substrate cost as well as therequirement for optical resolution of racemic intermediates.Accordingly, researchers have recently looked to bacterial or microbialsystems that produce enzymes useful in pantothenate biosynthesisprocesses (as bacteria are themselves capable of synthesizingpantothenate). In particular, bioconversion processes have beenevaluated as a means of favoring production of the preferred isomer ofpantothenic acid. Moreover, methods of direct microbial synthesis haverecently been examined as a means of facilitating D-pantothenateproduction.

There is still, however, significant need for improved pantothenateproduction processes, in particular, for microbial processes optimizedto produce higher yields of desired product.

SUMMARY OF THE INVENTION

The present invention relates to improved processes (e.g., microbialsyntheses) for the production of pantothenate. Pantothenate productionprocesses have been described in related applications which feature, forexample, microbes engineered to overexpress key enzymes of thepantothenate biosynthetic pathway and the isoleucine-valine biosyntheticpathway (see e.g., FIG. 1). Strains have been engineered that arecapable of producing >50 g/l of pantothenate in standard fermentationprocesses (see e.g., International Public. No. WO 01/21772 and U.S.Patent Application Ser. No. 60/262,995). In particular, increasing theexpression of the panB, panC, panD and panE1 genes and increasing theexpression of the ilvBNC and ilvD genes results in strains that convertglucose (pyruvate) to commercially attractive quantities ofpantothenate.

In order to enhance production levels of for example, pantothenate,various improvements on the above-described methods have now beendeveloped. For example, U.S. patent application Ser. No. 09/667,569describes production strains having modified (e.g., deleted ordecreased-activity) pantothenate kinase enzymes. In such strains thepantothenate levels are effectively increased by decreasing utilizationof pantothenate for coenzymeA (“CoA”) synthesis. U.S. Patent ApplicationSer. No. 60/262,995 further describes improved pantothenate-productionstrains that have been engineered to minimize utilization of variouspantothenate biosynthetic enzymes and/or isoleucine-valine biosyntheticenzymes and/or their respective substrates from being used to produce analternative product identified as[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMPA”).

The present invention features methods to further enhance pantothenateproduction by modulating a biosynthetic pathway that supplies asubstrate for the pantothenate biosynthetic pathway, namely themethylenetetrahydrofolate (“MTF”) biosynthetic pathway. In particular,it has been discovered that increasing levels of MTF by modification ofthe MTF biosynthetic pathway results in enhanced levels of the keypantothenate biosynthetic pathway intermediate, ketopantoate. Enhancedketopantoate levels, in turn, result in significantly enhancedpantothenate production levels in appropriately engineered stains. Inessence, the present inventors have identified a limiting step in theproduction of panto-compounds (e.g., pantothenate) by strains engineeredto overexpress, for example, the panB, panC, panD, panE1, ilvBNC andilvD genes, and describe herein a means for overcoming this limitationby modification of the MTF biosynthetic pathway.

At least three effective means of modifying the MTF biosynthetic pathwayare described herein. In one aspect, it has been demonstrated thatincreasing serine levels in the culture medium of pantothenate-producingmicroorganisms results in enhanced panto-compound production. It hasalso been demonstrated that increasing the synthesis or activity of3-phosphoglycerate dehydrogenase (the serA gene product), or thesynthesis or activity of serine hydroxymethyl transferase (the glyA geneproduct), thereby enhancing serine and methylenetetrahydrofolatebiosynthesis in appropriately engineered microorganisms, increasespanto-compound production. Increased synthesis of 3-phosphoglyceratedehydrogenase (the sera gene product) is achieved, for example, byoverexpressing sera from an appropriately-engineered expressioncassette. Increased synthesis of serine hydroxymethyl transferase (theglyA gene product) is achieved, for example, by overexpressing glyA froman appropriately-engineered expression cassette. Alternatively, levelsof serine hydroxymethyl transferase (the glyA gene product) areincreased by altering the regulation of the glyA gene. For example,mutation or deletion of the gene encoding a negative regulator (i.e.,repressor) of glyA expression, the purR gene, effectively increases glyAexpression. Additional methods suitable for increasing MTF levels inpanto-compound producing microoganisms involve deregulating enzymesresponsible for converting glycine to MTF (e.g., glycine cleavageenzymes).

Accordingly, in one aspect the invention features processes for theenhanced production of pantoate and pantothenate that involve culturingmicroorganisms having modified pantothenate biosynthetic enzymeactivities and having modified methylenetetrahydrofolate (MTF)biosynthetic enzyme activities under conditions such that pantothenateproduction is enhanced. In another aspect the invention featuresprocesses for the enhanced production of pantoate and pantothenate thatinvolve culturing microorganisms having modified pantothenatebiosynthetic enzyme activities, having modified isoleucine-valine (ilv)biosynthetic enzymes, and having modified methylenetetrahydrofolate(MTF) biosynthetic enzyme activities under conditions such thatpantothenate production is enhanced. In particular, the inventionfeatures methods for enhancing production of desired products (e.g.,pantoate and/or pantothenate) by increasing the levels of a keyintermediate, ketopantoate, by enzymes that contribute to its synthesis.Preferred methods result in production of pantothenate at levels greaterthan 50, 60, 70 or more g/L after 36 hours of culturing themicroorganisms, or such that at least 60, 70, 80, 90, 100, 110, 120 ormore g/L pantothenate is produced after 36 hours of culturing themicroorganisms. Recombinant microorganisms and conditions for culturingsame are also are featured. Also featured are compositions produced bysuch microorganisms.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the pantothenate andisoleucine-valine (ilv) biosynthetic pathways. Pantothenate biosyntheticenzymes are depicted in bold and their corresponding genes indicated initalics. Isoleucine-valine (ilv) biosynthetic enzymes are depicted inbold italics and their corresponding genes indicated in italics.

FIG. 2 is a schematic representation of the methylenetetrahydrofolate(“MTF”) biosynthetic pathway in E. coli (and presumably in B. subtilis).

FIG. 3 is a schematic representation of the construction of the plasmidpAN665.

FIG. 4 is a schematic representation of the construction of the plasmidpAN670.

FIG. 5 is a schematic representation of the plasmid pAN004.

FIG. 6 is a schematic representation of the plasmid pAN396.

FIG. 7 is a schematic representation of the plasmid pAN393.

FIG. 8 is a schematic representation of the structure of pAN835F, aclone of the B. subtilis purR gene.

FIG. 9 is a schematic representation of the structure of pAN838F, aplasmid designed to install a disruption of the B. subtilis purR gene.

FIG. 10 is a schematic representation of the structure of pAN821, aplasmid designed to delete a portion of the serA gene, selecting forkanamycin resistance.

FIG. 11 is a schematic representation of the structure of pAN824, aplasmid designed to integrate a non-amplifiable P₂₆serA cassette at thesera locus, selecting for Ser⁺.

FIG. 12 is a schematic representation of the structure of pAN395, amedium copy plasmid designed to integrate and amplify a P26 serAexpression cassette at the serA locus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to improved methods for producingpanto-compounds (e.g., ketopantoate, pantoate and/or pantothenate) andstrains engineered for use in said improved methods. Strains capable ofproducing >50 g/l of pantothenate can be constructed as taught inInternational Patent Application Serial No. WO 01/21772 and in U.S.Patent Application Ser. No. 60/262,995. By increasing the expression ofthe panB, panC, panD and panE1 genes and by increasing the expression ofthe ilvBNC and ilvD genes, one can design strains (e.g., Bacillusstrains) that convert glucose (pyruvate) to commercially attractivequantities of pantothenate.

However, it has now been discovered that in strains engineered toexpress high levels of the panB gene product, ketopantoatehydroxymethyltransferase (e.g., PAS824, described in U.S. patentapplication Ser. No. 09/667,569 and PA668-24, described in U.S. PatentApplication Ser. No. 60/262,995), a limiting step for further increasesin the production of pantothenate is still the conversion ofα-ketoisovalerate (α-KIV) to ketopantoate. Methods to increase thesynthesis of α-KIV were described previously in International PatentApplication Serial No. WO 01/21772 and U.S. Patent Application Ser. No.60/262,995. Here we disclose that even further increases in pantothenateproduction can be achieved by engineering panto-compound producingmicroorganisms such that the level of MTF, or the rate of MTF synthesisis enhanced or increased.

Accordingly, the present invention features methods for improvingpanto-compound production that involve modulating themethylenetetrahydrofolate (“MTF”) biosynthetic pathway. In particular,increasing of levels in panto-compound producing microbe is an effectivemeans of enhancing ketopantoate production, and in turn results inenhanced pantoate and/or pantothenate production inappropriately-engineered recombinant microorganisms.

Ketopantoate hydroxymethlylenetransferase catalyzes the production ofketopantoate from α-ketoisovalerate (“α-KIV”) and MTF (see e.g., FIG.1). In particular, the enzyme catalyzes the transfer of a hydroxymethylgroup from MTF to α-KIV to yield ketopantoate. Both α-KIV and MTF aresubstrates for this reaction, and their syntheses can be increased inorder to improve production of ketopantoate. The pathway for MTFbiosynthesis in E. coli (and also in Bacillus subtilis) is outlined inFIG. 2. MTF is synthesized from tetrahydrofolate and serine in areaction catalyzed by the glyA gene that encodes serine hydroxymethyltransferase. For improved MTF synthesis the cells need increasedquantities of both substrates and the product of the glyA gene.

In one embodiment the invention features processes for the enhancedproduction of pantothenate that involve culturing a microorganism having(i) a deregulated pantothenate biosynthetic pathway (e.g., having one,two, three or four pantothenate biosynthetic enzymes deregulated) and(ii) a deregulated methylenetethrhydrofolate (MTF) biosynthetic pathway(e.g., having at least one or two MTF biosynthetic enzymes deregulated),under conditions such that pantothenate production is enhanced.Exemplary pantothenate biosynthetic enzymes include ketopantoatehydroxymethyltransferase, ketopantoate reductase, pantothenatesynthetase and aspartate-α-decarboxylase. Exemplary MTF biosyntheticenzymes include the serA gene product and the glyA gene product.

In another embodiment, the invention features processes for the enhancedproduction of pantothenate that involve culturing a microorganism having(i) a deregulated pantothenate biosynthetic pathway (e.g., having one,two, three or four pantothenate biosynthetic enzymes deregulated), (ii)a deregulated isoleucine-valine (ilv) biosynthetic pathway (e.g., havingone, two or three ilv biosynthetic enzymes deregulated), and (iii) aderegulated MTF biosynthetic pathway (e.g., having at least one or twoMTF biosynthetic enzymes deregulated), under conditions such thatpantothenate production is enhanced. Exemplary ilv biosynthetic enzymesinclude acetohydroxyacid acid synthetase, acetohydroxyacidisomeroreductase, and dihydroxyacid dehydratase.

In another embodiment, the invention features processes for theproduction of pantothenate that involve culturing a microorganism havinga deregulated pantothenate biosynthetic pathway, a deregulated ilvbiosynthetic pathway, and a deregulated MTF biosynthetic pathway, suchthat at least 50 g/L pantothenate is produced after 36 hours ofculturing the microorganism, preferably such that at least 60 g/Lpantothenate is produced after 36 hours of culturing the microorganism,more preferably such that at least 70 g/L pantothenate is produced after36 hours of culturing the microorganism, and most preferably such thatat least 80 g/L pantothenate, at least 90 g/L pantothenate at least 100g/L pantothenate, at least 110 g/L pantothenate, or at least 120 g/Lpantothenate (or more) is produced after 36 hours of culturing themicroorganism.

In another embodiment, the invention features processes for theproduction of pantothenate that involve culturing a microorganism havinga deregulated pantothenate biosynthetic pathway, a deregulated ilvbiosynthetic pathway, and a deregulated MTF biosynthetic pathway,deregulated such that at least 70 g/L pantothenate is produced after 48hours of culturing the microorganism, preferably such that at least 80g/L pantothenate is produced after 48 hours of culturing themicroorganism, and more preferably such that at least 90 g/Lpantothenate is produced after 48 hours of culturing the microorganism.

In one exemplary embodiment, deregulation of the MTF biosyntheticpathway is achieved by deregulating the serA gene product in apanto-compound producing strain, for example, by expressing the serAgene constitutively or by introducing a feedback resistant allele ofserA. In another exemplary embodiment, deregulation of the MTFbiosynthetic pathway is achieved by deregulating the glyA gene productin a panto-compound producing strain, for example, by overexpressing theglyA gene or modulating repression of the glyA gene by mutating ordisrupting the purR gene product. In other exemplary embodiments, MTFbiosynthesis is modulated by increasing serine in the culture medium orderegualting glycine cleavage enzymes.

The invention further features methods as described above, whereinpantothenate production is further enhanced by regulating pantothenatekinase activity (e.g., wherein pantothenate kinase activity isdecreased). In one embodiment, CoaA is deleted and CoaX isdownregulated. In another embodiment, CoaX is deleted and CoaA isdownregulated. In yet another embodiment, CoaX and CoaA aredownregulated. The invention further features methods as describedabove, wherein the microorganisms are cultured under conditions ofexcess serine. The invention further features methods as describedabove, wherein the microorganisms have the pantothenate biosyntheticpathway deregulated such that pantothenate production is independent ofβ-alanine feed.

Products synthesized according to the processes of the invention arealso featured, as are compositions that include pantothenate producedaccording to said processes. Recombinant microorganisms for use in theprocesses of the invention are also featured. In one embodiment, theinvention features a recombinant microorganism for the enhancedproduction of pantothenate having a deregulated pantothenatebiosynthetic pathway and a deregulated MTF biosynthetic pathway. Inanother embodiment, the invention features a recombinant microorganismfor the enhanced production of pantothenate having a deregulatedpantothenate biosynthetic pathway, a deregulated MTF biosyntheticpathway and a deregulated ilv pathway. Microorganisms can flier havereduced pantothenate kinase activity. Preferred microorganisms belong tothe genus Bacillus, for example Bacillus subtilis.

As described above, certain aspects of the invention, feature processesfor the enhanced production of panto-compounds (e.g., pantoate and/orpantothenate) that involve culturing microorganisms having at least aderegulated pantothenate biosynthetic pathway. The term “pantothenatebiosynthetic pathway” includes the biosynthetic pathway involvingpantothenate biosynthetic enzymes (e.g., polypeptides encoded bybiosynthetic enzyme-encoding genes), compounds (e.g., substrates,intermediates or products), cofactors and the like utilized in theformation or synthesis of pantothenate. The term “pantothenatebiosynthetic pathway” includes the biosynthetic pathway leading to thesynthesis of pantothenate in microorganisms (e.g., in vivo) as well asthe biosynthetic pathway leading to the synthesis of pantothenate invitro.

As used herein, a microorganism “having a deregulated pantothenatebiosynthetic pathway” includes a microorganism having at least onepantothenate biosynthetic enzyme deregulated (e.g., overexpressed) (bothterms as defined herein) such that pantothenate production is enhanced(e.g., as compared to pantothenate production in said microorganismprior to deregulation of said biosynthetic enzyme or as compared to awild-type microorganism). The term “pantothenate” includes the free acidform of pantothenate, also referred to as “pantothenic acid” as well asany salt thereof (e.g., derived by replacing the acidic hydrogen ofpantothenate or pantothenic acid with a cation, for example, calcium,sodium, potassium, ammonium, magnesium), also referred to as a“pantothenate salt.” The term “pantothenate” also includes alcoholderivatives of pantothenate. Preferred pantothenate salts are calciumpantothenate or sodium pantothenate. A preferred alcohol derivative ispantothenol. Pantothenate salts and/or alcohols of the present inventioninclude salts and/or alcohols prepared via conventional methods from thefree acids described herein. In another embodiment, a pantothenate saltis synthesized directly by a microorganism of the present invention. Apantothenate salt of the present invention can likewise be converted toa free acid form of pantothenate or pantothenic acid by conventionalmethodology. The term “pantothenate” is also abbreviated as “pan”herein.

Preferably, a microorganism shaving a deregulated pantothenatebiosynthetic pathway includes a microorganism having at least onepantothenate biosynthetic enzyme deregulated (e.g., overexpressed) suchthat pantothenate production is 1 g/L or greater. More preferably, amicroorganism “having a deregulated pantothenate biosynthetic pathway”includes a microorganism having at least one pantothenate biosyntheticenzyme deregulated (e.g., overexpressed) such that pantothenateproduction is 2 g/L or greater. Even more preferably, a microorganism“having a deregulated pantothenate biosynthetic pathway” includes amicroorganism having at least one pantothenate biosynthetic enzymederegulated (e.g., overexpressed) such that pantothenate production is10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g 60 g/L, 70 g/L, 80 g/L, 90 g/L, orgreater.

The term “pantothenate biosynthetic enzyme” includes any enzyme utilizedin the formation of a compound (e.g., intermediate or product) of thepantothenate biosynthetic pathway. For example, synthesis of pantoatefrom α-ketoisovalerate (α-KIV) proceeds via the intermediate,ketopantoate. Formation of ketopantoate is catalyzed by the pantothenatebiosynthetic enzyme PanB or ketopantoate hydroxymethyltransferase (thepanB gene product). Formation of pantoate is catalyzed by thepantothenate biosynthetic enzyme PanE1 or ketopantoate reductase (thepanE1 gene product). Synthesis of β-alanine from aspartate is catalyzedby the pantothenate biosynthetic enzyme PanD oraspartate-α-decarboxylase (the panD gene product). Formation ofpantothenate from pantoate and β-alanine (e.g., condensation) iscatalyzed by the pantothenate biosynthetic enzyme PanC or pantothenatesynthetase (the panC gene product). Pantothenate biosynthetic enzymesmay also perform an alternative function as enzymes in the HMBPAbiosynthetic pathway described herein.

Accordingly, in one embodiment, the invention features a process for theenhanced production of pantothenate that includes culturing amicroorganism having at least one pantothenate biosynthetic enzymederegulated (e.g., deregulated such that pantothenate production isenhanced), said enzyme being selected, for example, from the groupconsisting of PanB (or ketopantoate hydroxymethyltransferase), PanC (orpantothenate synthetase), PanD (or aspartate-α-decarboxylase), PanE1 (orketopantoate reductase). In another embodiment, the invention features aprocess for the enhanced production of pantothenate that includesculturing a microorganism having at least two pantothenate biosyntheticenzymes deregulated, said enzymes being selected, for example, from thegroup consisting of PanB (or ketopantoate hydroymethyltransferase), PanC(or pantothenate synthetase), PanD (or aspartate-α-decarboxylase), andPanE1 (or ketopantoate reductase). In another embodiment, the inventionfeatures a process for the enhanced production of pantothenate thatincludes culturing a microorganism having at least three pantothenatebiosynthetic enzymes deregulated, said enzymes being selected, forexample, from the group consisting of PanB (or ketopantoatehydroxymethyltransferase), PanC (or pantothenate synthetase) PanD (oraspartate-α-decarboxylase), and PanE1 (or ketopantoate reductase). Inanother embodiment, the invention features a process for the enhancedproduction of pantothenate that includes culturing a microorganismhaving at least four pantothenate biosynthetic enzymes deregulated, forexample, a microorganism having PanB (orketopantoate-α-hydroxymethyltransferase), PanC (or pantothenatesynthetase), PanD (or aspartate decarboxylase), and PanE1 (orketopantoate reductase) deregulated.

In another aspect, the invention features processes for the enhancedproduction of pantothenate that involve culturing microorganisms havinga deregulated isoleucine-valine biosynthetic pathway. The term“isoleucine-valine biosynthetic pathway” includes the biosyntheticpathway involving isoleucine-valine biosynthetic enzymes (e.g.,polypeptides encoded by biosynthetic enzyme-encoding genes), compounds(e.g., substrates, intermediates or products), cofactors and the likeutilized in the formation or synthesis of conversion of pyruvate tovaline or isoleucine. The term “isoleucine-valine biosynthetic pathway”includes the biosynthetic pathway leading to the synthesis of valine orisoleucine in microorganisms (e.g., in vivo) as well as the biosyntheticpathway leading to the synthesis of valine or isoleucine in vitro.

As used herein, a microorganism “having a deregulated isoleucine-valine(ilv) pathway” includes a microorganism having at least oneisoleucine-valine (ilv) biosynthetic enzyme deregulated (e.g.,overexpressed) (both terms as defined herein) such that isoleucineand/or valine and/or the valine precursor, α-ketoisovaerate (α-KIV)production is enhanced (e.g., as compared to isoleucine and/or valineand/or α-KIV production in said microorganism prior to deregulation ofsaid biosynthetic enzyme or as compared to a wild-type microorganism).FIG. 1 includes a schematic representation of the isoleucine-valinebiosynthetic pathway. Isoleucine-valine biosynthetic enzymes aredepicted in bold italics and their corresponding genes indicated initalics. The term “isoleucine-valine biosynthetic enzyme” includes anyenzyme utilized in the formation of a compound (e.g., intermediate orproduct) of the isoleucine-valine biosynthetic pathway. According toFIG. 1, synthesis of valine from pyruvate proceeds via theintermediates, acetolactate, α,βdihydroxyisovalerate (α,β-DHIV andα-ketoisovalerate (α-KIV). Formation of acetolactate from pyruvate iscatalyzed by the isoleucine-valine biosynthetic enzyme acetohydroxyacidsynthetase (the ilvBN gene products, or alternatively, the alsS geneproduct). Formation of α,β-DHIV from acetolactate is catalyzed by theisoleucine-valine biosynthetic enzyme acetohydroxyacid isomeroreductase(the ilvC gene product). Synthesis of α-KIV from α,β-DHIV is catalyzedby the isoleucine-valine biosynthetic enzyme dihydroxyacid dehydratase(the ilvD gene product). Moreover, valine and isoleucine can beinterconverted with their respective α-keto compounds by branched chainamino acid transaminases. Isoleucine-valine biosynthetic enzymes mayalso perform an alternative function as enzymes in the HMBPAbiosynthetic pathway described herein.

Accordingly, in one embodiment, the invention features a process for theenhanced production of pantothenate that includes culturing amicroorganism having at least one isoleucine-valine (ilv) biosyntheticenzyme deregulated (e.g., deregulated such that valine and/or isoleucineand/or α-KIV production, is enhanced) said enzyme being selected, forexample, from the group consisting of IlvBN, AlsS (or acetohydroxyacidsynthetase), IlvC; (or acetohydroxyacid isomeroreductase) and IlvD (ordihydroxyacid dehydratase). In another embodiment, the inventionfeatures a process for the enhanced production of pantothenate thatincludes culturing a microorganism having at least two isoleucine-valine(ilv) biosynthetic enzymes deregulated, said enzyme being selected, forexample, from the group consisting of IlvBN, AlsS (or acetohydroxyacidsynthetase), IlvC (or acetohydroxyacid isomeroreductase) and IlvD (ordihydroxyacid dehydratase). In another embodiment, the inventionfeatures a process for the enhanced production of pantothenate thatincludes culturing a microorganism having at least threeisoleucine-valine (ilv) biosynthetic enzymes deregulated, for example,said microorganism having IlvBN or AlsS (or acetohydroxyacidsynthetase), IlvC (or acetohydroxyacid isomeroreductase) and IlvD (ordihydroxyacid dehydratase) deregulated.

As mentioned herein, enzymes of the pantothenate biosynthetic pathwayand/or the isoleucine-valine (ilv) pathway have been discovered to havean alternative activity in the synthesis of[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”) or the[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”)biosynthetic pathway. The term“[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (“HMBPA”)biosynthetic pathway” includes the alternative biosynthetic pathwayinvolving biosynthetic enzymes and compounds (e.g., substrates and thelike) traditionally associated with the pantothenate biosyntheticpathway and/or isoleucine-valine (ilv) biosynthetic pathway utilized inthe formation or synthesis of HMBPA. The term “HMBPA biosyntheticpathway” includes the biosynthetic pathway leading to the synthesis ofHMBPA in microorganisms (e.g., in vivo) as well as the biosyntheticpathway leading to the synthesis of HMBPA in vitro.

The term “HMBPA” biosynthetic enzyme includes any enzyme utilized in theformation of a compound (e.g., intermediate or product) of the HMBPAbiosynthetic pathway. For example, synthesis of 2-hydroxyisovaleric acid(α-HIV) from α-ketoisovalerate (α-KIV) is catalyzed by the panE1 orpanE2 gene product (PanE1 is alternatively referred to herein asketopantoate-reductase) and/or is catalyzed by the ilvC gene product(alternatively referred to herein as acetohydroxyacid isomeroreductase).Formation of HMBPA from β-alanine and α-HIV is catalyzed by the panCgene product (alternatively referred to herein as pantothenatesynthetase).

The term “[R]-3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid(“HMBPA”)” includes the free acid form of HMBPA, also referred to as“[R]-3-(2-hydroxy-3-methyl-butyrlamino)-propionate” as well as any saltthereof (e.g., derived by replacing the acidic hydrogen of3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid or3-(2-hydroxy-3-methyl-butyrylamino)-propionate with a cation, forexample, calcium, sodium, potassium, ammonium, magnesium), also referredto as a “3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid salt” or“HMBPA salt.” Preferred HMBPA salts are calcium HMBPA or sodium HMBPA.HMBPA salts of the present invention include salts prepared viaconventional methods from the free acids described herein. An HMBPA saltof the present invention can likewise be converted to a free acid formof 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid or3-(2-hydroxy-3-methyl-butyrylamino)-propionate by conventionalmethodology.

In preferred embodiments, the invention features processes for theenhanced production of panto-compounds (e.g., pantoate and/orpantothenate) that involve culturing a microorganism having aderegulated methylenetetrahydrofolate (MTF) biosynthetic pathway. Theterm “methylenetetrahydrofolate (MTF) biosynthetic pathway” refers tothe biosynthetic pathway involving MTF biosynthetic enzymes (e.g.,polypeptides encoded by biosynthetic enzyme-encoding genes), compounds(e.g., substrates, intermediates or products), cofactors and the likeutilized in the formation or synthesis of the PanB substrate, MTF. Theterm “methylenetetrahydrofolate MTF) biosynthetic pathway” refers to thebiosynthetic pathway leading to the synthesis of MTF in vivo (e.g., thepathway in E. coli, as depicted in FIG. 2) as well as the biosyntheticpathway leading to the synthesis of MTF in vitro. The term“methylenetetrahydrofolate (MTF) biosynthetic enzyme” includes anyenzyme utilized in the formation of a compound (e.g., intermediate orproduct) of the methylenetetrahydrofolate (MTF) biosynthetic pathway.

The present invention is based, at least in part, on the discovery thatderegulation of certain MTF biosynthetic enzymes results in enhancedproduction of MTF. A MTF biosynthetic enzyme, the deregulation of whichresults in enhanced MTF production, is termed a “MTFbiosynthesis-enhancing enzyme”. Exemplary “MTF biosynthesis-enhancingenzymes” are the serA gene product (3-phosphoglycerate dehydrogenase)and the glyA gene product (serine hydroxymethyl transferase). Amicroorganism “having a deregulated methylenetetrahydrofolate (MTF)biosynthetic pathway”, is a microorganism having at least one MTFbiosynthesis-enhancing enzyme deregulated (e.g., overexpressed) suchthat MTF production or biosynthesis is enhanced (e.g., as compared toMTF production in said microorganism prior to deregulation of saidbiosynthetic enzyme or as compared to a wild type microorganism).

In one embodiment, the invention features a process for the enhancedproduction of panto-compounds (e.g., pantoate and/or pantothenate) thatincludes culturing a microorganism having a deregulated“methylenetetrahydrofolate (MTF) biosynthetic pathway”, as definedherein. In another embodiment, the invention features a process for theenhanced production of panto-compounds (e.g., pantoate and/orpantothenate) that includes culturing a microorganism having aderegulated MTF biosynthesis-enhancing enzyme. In preferred embodiments,the invention features processes for the enhanced production ofpanto-compounds (e.g. pantoate and/or pantothenate) that includesculturing a microorganism having a deregulated glyA gene product (serinehydroxymethyl transferase) and/or a deregulated serA gene product(3-phosphoglycerate dehydrogenase).

Yet another aspect of the present invention features processes for theenhanced production of pantothenate that include culturingmicroorganisms under culture conditions selected to favor pantothenateproduction, for example, by culturing microorganisms with excess serine(a glyA substrate) in the medium. The term “excess serine” includesserine levels increased or higher that those routinely utilized forculturing the microorganism in question. For example, culturing theBacillus microorganisms described in the instant Examples is routinelydone in the presence of about 0-2.5 g/L serine. Accordingly, excessserine levels can include levels of greater than 2.5 g/L serine, forexample, between about 2.5 and 10 g/L serine. Excess serine levels caninclude levels of greater than 5 g/L serine, for example, between about5 and 10 g/L serine.

Yet another aspect of the present invention features culturing themicroorganisms described herein under conditions such that pantothenateproduction is further increased, for example, by increasing pantothenateand/or isoleucine-valine (ilv) biosynthetic pathway precursors and/orintermediates as defined herein (e.g., culturing microorganisms in thepresence of excess β-alanine, valine and/or α-KIV) or, alternatively,further modifying said microorganisms such that they are capable ofproducing significant levels of β-alanine in the absence of a β-alaninefeed (i.e., β-alanine independent microorganisms, as described in U.S.patent application Ser. No. 09/667,569).

Yet another aspect of the invention features further regulatingpantothenate kinase activity in pantothenate-producing strains such thatpantothenate production is enhanced. Pantothenate kinase is a key enzymecatalyzing the formation of Coenzyme A (CoA) from pantothenate (seee.g., U.S. patent application Ser. No. 09/09/667,569). Regulation ofpantothenate kinase (e.g., decreasing the activity or level ofpantothenate kinase) reduces the production of CoA, favoringpantothenate accumulation. In one embodiment, pantotheante kinaseactivity is decreased by deleting CoaA and downregulating CoaX activity(CoaA and CoaX are both capable of catalyzing the first step in CoAbiosynthesis in certain microorganisms). In another embodiment,pantothenate kinase activity is decreased by deleting CoaX anddownregulating CoaA. In yet another embodiment, pantotheante kinaseactivity is decreased by downregulating CoaA and CoaX activities.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Targeting Genes Encoding Various Pantothenate and/orIsoleucine-Valine (ilv) and/or Methylenetetrahydrofolate (MTF)Biosynthetic Enzymes

In one embodiment, the present invention features modifying orincreasing the level of various biosynthetic enzymes of the pantothenateand/or isoleucine-valine (ilv) and/or methylenetetrahydrofolate (MTF)biosynthetic pathways. In particular, the invention features modifyingvarious enzymatic activities associated with said pathways by modifyingor altering the genes encoding said biosynthetic enzymes.

The term “gene”, as used herein, includes a nucleic acid molecule (e.g.,a DNA molecule or segment thereof) that, in an organism, can beseparated from another gene or other genes, by intergenic DNA (i.e.,intervening or spacer DNA which naturally flanks the gene and/orseparates genes in the chromosomal DNA of the organism). Alternatively,a gene may slightly overlap another gene (e.g., the 3′ end of a firstgene overlapping the 5′ end of a second gene), the overlapping genesseparated from other genes by intergenic DNA. A gene may directsynthesis of an enzyme or other protein molecule (e.g., may comprisecoding seqeunces, for example, a contiguous open reading frame (ORF)which encodes a protein) or may itself be functional in the organism. Agene in an organism, may be clustered in an operon, as defined herein,said operon being separated from other genes and/or operons by theintergenic DNA. An “isolated gene”, as used herein, includes a genewhich is essentially free of sequences which naturally flank the gene inthe chromosomal DNA of the organism from which the gene is derived(i.e., is free of adjacent coding sequences that encode a second ordistinct protein, adjacent structural sequences or the like) andoptionally includes 5′ and 3′ regulatory sequences, for example promotersequences and/or terminator, sequences. In one embodiment, an isolatedgene includes predominantly ceding sequences for a protein (e.g.,sequences which encode Bacillus proteins). In another embodiment, anisolated gene includes coding sequences for a protein (e.g., for aBacillus protein)- and adjacent 5′ and/or 3′ regulatory sequences fromthe chromosomal DNA of the organism from which the gene is derived(e.g., adjacent 5′ and/or 3′ Bacillus regulatory sequences). Preferably,an isolated gene contains less than about 10 kb, 5 kb, 2 kb, 1 kb, 0.5kb, 0.2 kb, 0.1 kb, 50 bp, 25 bp or 10 bp of nucleotide sequences whichnaturally flank the gene in the chromosomal DNA of the organism fromwhich the gene is derived.

The term “operon” includes at least two adjacent genes or ORFs,optionally overlapping in sequence at either the 5′ or 3′ end of atleast one gene or ORF. The term “operon” includes a coordinated unit ofgene expression that contains a promoter and possibly a regulatoryelement associated with one or more adjacent genes or ORFs (e.g.,structural genes encoding enzymes, for example, biosynthetic enzymes).Expression of the genes (e.g., structural genes) can be coordinatelyregulated, for example, by regulatory proteins binding to the regulatoryelement or by anti-termination of transcription. The genes of an operon(e.g., structural genes) can be transcribed to give a single mRNA thatencodes all of the proteins.

A “gene having a mutation” or “mutant gene” as used herein, includes agene having a nucleotide sequence which includes at least one alteration(e.g., substitution, insertion, deletion) such that the polypeptide orprotein encoded by said mutant exhibits an activity that differs fromthe polypeptide or protein encoded by the wild-type nucleic acidmolecule or gene. In one embodiment, a gene having a mutation or mutantgene encodes a polypeptide or protein having an increased activity ascompared to the polypeptide or protein encoded by the wild-type gene,for example, when assayed under similar conditions (e.g., assayed inmicroorganisms, cultured at the same temperature). As used herein, an“increased activity” or “increased-enzymatic activity” is one that is atleast 5% greater than that of the polypeptide or protein encoded by thewild-type nucleic acid molecule or gene, preferably at least 5-10%greater, more preferably at least 10-25% greater and even morepreferably at least 25-50%, 50-75% or 75-100% greater than that of thepolypeptide or protein encoded by the wild-type nucleic acid molecule orgene. Ranges intermediate to the above-recited values, e.g., 75-85%,85-90%, 90-95%, are also intended to be encompassed by the presentinvention. As used herein, an “increased activity” or “increasedenzymatic activity” can also include an activity that is at least1.25-fold greater than the activity of the polypeptide or proteinencoded by the wild-type gene, preferably at least 1.5-fold greater,more preferably at least 2-fold greater and even more preferably atleast 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-foldgreater than the activity of the polypeptide or protein encoded by thewild type gene.

In another embodiment, a gene having a mutation, or mutant gene encodesa polypeptide or protein having a reduced activity as compared to thepolypeptide or protein encoded by the wild-type gene, for example, whenassayed under similar conditions (e.g., assayed in microorganismscultured at the same temperature). A mutant gene also can encode nopolypeptide or have a reduced level of production of the wild-typepolypeptide. As used herein, a “reduced activity” or “reduced enzymaticactivity” is one that is at least 5% less than that of the polypeptideor protein encoded by the wild-type nucleic acid molecule or gene,preferably at least 5-10% less, more preferably at least 10-25% less andeven more preferably at least 25-50%, 50-75% or 75-100% less than thatof the polypeptide or protein encoded by the wild-type nucleic acidmolecule or gene. Ranges intermediate to the above-recited values, e.g.,75-85%, 85-90%, 90-95%, are also intended to be encompassed by thepresent invention. As used herein, a “reduced activity” or “reducedenzymatic activity” can also include an activity that has been deletedor “knocked out” (e.g., approximately 100% less activity than that ofthe polypeptide or protein encoded by the wild-type nucleic acidmolecule or gene).

Activity can be determined according to any well accepted assay formeasuring activity of a particular protein of interest. Activity can bemeasured or assayed directly, for example, measuring an activity of aprotein in a crude cell extract or isolated or purified from a cell ormicroorganism. Alternatively, an activity can be measured or assayedwithin a cell or microorganism or in an extracellular medium. Forexample, assaying for a mutant gene (i.e., said mutant encoding areduced enzymatic activity) can be accomplished by expressing themutated gene in a microorganism, for example, a mutant microorganism inwhich the enzyme is a temperature sensitive, and assaying the mutantgene for the ability to complement a temperature sensitive (Ts) mutantfor enzymatic activity. A mutant gene that encodes an “increasedenzymatic activity” can be one that complements the Ts mutant moreeffectively than, for example, a corresponding wild-type-gene. A mutantgene that encodes a “reduced enzymatic activity” is one that complementsthe Ts mutant less-effectively than, for example, a correspondingwild-type gene.

It will be appreciated by the skilled artisan that even a singlesubstitution in a nucleic acid or gene sequence (e.g., a basesubstitution that encodes an amino acid change in the correspondingamino acid sequence) can dramatically affect the activity of an encodedpolypeptide or protein as compared to the corresponding wild-typepolypeptide or protein. A mutant gene (e.g., encoding a mutantpolypeptide or protein), as defined herein, is readily distinguishablefrom a nucleic acid or gene encoding a protein homologue in that amutant gene encodes a protein or polypeptide having an altered activity,optionally observable as a different or distinct phenotype in amicroorganism expressing said mutant gene or producing said mutantprotein or polypeptide (i.e., a mutant microorganism) as compared to acorresponding microorganism expressing the wild-type gene. By contrast,a protein homologue can have an identical or substantially similaractivity, optionally phenotypically indiscernable when produced in amicroorganism, as compared to a corresponding microorganism expressingthe wild-type gene. Accordingly it is not, for example, the degree ofsequence identity between nucleic acid molecules, genes, protein orpolypeptides that serves to distinguish between homologues and mutants,rather it is the activity of the encoded protein or polypeptide thatdistinguishes between homologues and mutants: homologues having, forexample, low (e.g., 30-50% sequence identity) sequence identity yethaving substantially equivalent functional activities, and mutants, forexample sharing 99% sequence identity yet having dramatically differentor altered functional activities.

It will also be appreciated by the skilled artisan that nucleic acidmolecules, genes, protein or polypeptides for use in the instantinvention can be derived from any microorganisms having a MTFbiosynthetic pathway, an ilv biosynthetic pathway or a pantothenatebiosynthetic pathway. Such nucleic acid molecules, genes, protein orpolypeptides can be identified by the skilled artisan using knowntechniques such as homology screening, sequence comparison and the like,and can be modified by the skilled artisan in such a way that expressionor production of these nucleic acid molecules, genes, protein orpolypeptides occurs in a recombinant microorganism (e.g., by usingappropriate promotors, ribosomal binding sites, expression orintegration vectors, modifying the sequence of the genes such that thetranscription is increased (taking into account the preferable codonusage), etc., according to techniques described herein and those knownin the art).

In one embodiment, the genes of the present invention are derived from aGram positive microorganism organism (e.g., a microorganism whichretains basic dye, for example, crystal violet, due to the presence of aGram-positive wall surrounding the microorganism). The term “derivedfrom” (e.g., “derived from” a Gram positive microorganism) refers to agene which is naturally found in the microorganism (e.g., is naturallyfound in a Gram positive microorganism). In a preferred embodiment, thegenes of the present invention are derived from a microorganismbelonging to a genus selected from the group consisting of Bacillus,Cornyebacterium (e.g., Cornyebacterium glutamicum), Lactobacillus,Lactococci and Streptomyces. In a more preferred embodiment, the genesof the present invention are derived from a microorganism is of thegenus Bacillus. In another preferred embodiment, the genes of thepresent invention are derived from a microorganism selected from thegroup consisting of Bacillus subtilis, Bacillus lentimorbus, Bacilluslentus, Bacillus firmus, Bacillus pantothenticus, Bacillusamyloliquefaciens, Bacillus cereus, Bacillus circulans, Bacilluscoagulans, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus thuringieisis, Bacillus halodurans, and other Group 1Bacillus species, for example, as characterized by 16S rRNA type. Inanother preferred embodiment, the gene is derived from Bacillus brevisor Bacillus stearothermophilus. In another preferred embodiment, thegenes of the present invention are derived from a microorganism selectedfrom the group consisting of Bacillus licheniformis, Bacillusamyloliquefaciens, Bacillus subtilis, and Bacillus pumilus. In aparticularly preferred embodiment, the gene is derived from Bacillussubtilis(e.g., is Bacillus subtilis-derived). The term “derived fromBacillus subtilis” or “Bacillus subtilis-derived” includes a gene whichis naturally found in the microorganism Bacillus subtilis. Includedwithin the scope of the present invention are Bacillus-derived genes(e.g., B. subtilis-derived genes), for example, Bacillus or B. subtilispurR genes, serA genes, glyA genes, coax genes, coaA genes, pan genesand/or ilv genes.

In another embodiment, the genes of the present invention are derivedfrom a Gram negative (excludes basic dye) microorganism. In a preferredembodiment, the genes of the present invention are derived from amicroorganism belonging to a genus selected from the group consisting ofSalmonella (e.g., Salmonella typhimurium), Escherichia, Klebsiella,Serratia, and Proteus. In a more preferred embodiment, the genes of thepresent invention are derived from a microorganism of the genusEscherichia. In an even more preferred embodiment, the genes of thepresent invention are derived from Escherichia coli. In anotherembodiment, the genes of the present invention are derived fromSaccharomyces(e.g., Saccharomyces cerevisiae).

II. Recombinant Nucleic Acid Molecules and Vectors

The present invention further features recombinant nucleic acidmolecules (e.g., recombinant DNA molecules) that include genes describedherein (e.g., isolated genes), preferably Bacillus genes, morepreferably Bacillus subtilis, genes even more preferably Bacillussubtilis pantothenate biosynthetic genes and/or isoleucine-valine (ilv)biosynthetic genes and/or methylenetetrahydrofolate (MIT) biosyntheticgenes. The term “recombinant nucleic acid molecule” includes a nucleicacid molecule (e.g., a DNA molecule) that has been altered, modified orengineered such that it differs in nucleotide sequence from the nativeor natural nucleic acid molecule from which the recombinant nucleic acidmolecule was derived (e.g., by addition, deletion or substitution of oneor more nucleotides). Preferably, a recombinant nucleic acid molecule(e.g., a recombinant DNA molecule) includes an isolated gene of thepresent invention operably linked to regulatory sequences. The phrase“operably linked to regulatory sequence(s)” means that the nucleotidesequence of the gene of interest is linked to the regulatory sequence(s)in a manner which allows for expression (e.g., enhanced, increased,constitutive, basal, attenuated, decreased or repressed expression) ofthe gene, preferably expression of a gene product encoded by the gene(e.g., when the recombinant nucleic acid molecule is included in arecombinant vector, as defined herein, and is introduced into amicroorganism).

The term “regulatory sequence” includes nucleic acid sequences whichaffect (e.g., modulate or regulate) expression of other nucleic acidsequences (i.e., genes). In one embodiment, a regulatory sequence isincluded in a recombinant nucleic acid molecule in a similar oridentical position and/or orientation relative to a particular gene ofinterest as is observed for the regulatory sequence and gene of interestas it appears in nature, e.g., in a native position and/or orientation.For example, a gene of interest can be included in a recombinant nucleicacid molecule operably linked to a regulatory sequence which accompaniesor is adjacent to the gene of interest in the natural organism (e.g.,operably linked to “native” regulatory sequences (e.g., to the “native”promoter). Alternatively, a gene of interest can be included in arecombinant nucleic acid molecule operably linked to a regulatorysequence which accompanies or is adjacent to another (e.g., a different)gene in the natural organism. Alternatively, a gene of interest can beincluded in a recombinant nucleic acid molecule operably linked to aregulatory sequence from another organism. For example, regulatorysequences from other microbes (e.g., other bacterial regulatorysequences, bacteriophage regulatory sequences and the like) can beoperably linked to a particular gene of interest.

In one embodiment, a regulatory sequence is a non-native ornon-naturally-occurring sequence (e.g., a sequence which has beenmodified, mutated, substituted, derivatized, deleted including sequenceswhich are chemically synthesized). Preferred regulatory sequencesinclude promoters, enhancers, termination signals, anti-terminationsignals and other expression control elements (e.g., sequences to whichrepressors or inducers bind and/or binding sites for transcriptionaland/or translational regulatory proteins, for example, in thetranscribed mRNA). Such regulatory sequences are described, for example,in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Regulatorysequences include those which direct constitutive expression of anucleotide sequence in a microorganism (e.g., constitutive promoters andstrong constitutive promoters), those which direct inducible expressionof a nucleotide sequence in a microorganism (e.g., inducible promoters,for example, xylose inducible promoters) and those which attenuation orrepress expression of a nucleotide sequence in a microorganism (e.g.,attenuation signals or repressor binding sequences, for example, a PurRbinding site). It is also within the scope of the present invention toregulate expression of a gene of interest by removing or deletingregulatory sequences, for example, sequences involved in the negativeregulation of transcription can be removed such that expression of agene of interest is enhanced.

In one embodiment, a recombinant nucleic acid molecule of the presentinvention includes a nucleic acid sequence or gene that encodes at leastone-bacterial gene product (e.g., a pantothenate biosynthetic enzyme, anisoleucine-valine biosynthetic enzyme and/or a methylenetetrahydrofolate(MTF) biosynthetic enzyme) operably linked to a promoter or promotersequence. Preferred promoters of the present invention include Bacilluspromoters and/or bacteriophage promoters (e.g., bacteriophage whichinfect Bacillus). In one embodiment, a promoter is a Bacillus promoter,preferably a strong Bacillus promoter (e.g., a promoter associated witha biochemical housekeeping gene in Bacillus or a promoter associatedwith a glycolytic pathway gene in Bacillus). In another embodiment, apromoter is a bacteriophage promoter. In a preferred embodiment, thepromoter is from the bacteriophage SP01. In a particularly preferredembodiment, a promoter is selected from the group consisting of P₁₅, P₂₆or P_(veg), having for example, the following respective seqeunces:GCTATTGACGACAGCTATGGTTCACTGTCCACCAACCAAAACTGTGCTCAGTACCGCCAATATTTCTCCCTGAGGGGTACAAAGAGGTGTCCCTAGAAGAGATCCACGCTGTGTAAAAATTTTACAAAAAGGTATTGACTTTCCCTACAGGGTGTGTAATAATTTAATTACAGGCGGGGGCAACCCCGCCTGT (SEQ ID. NO: 1),GCCTACCTAGCTTCCAAGAAAGATATCCTAACAGCACAAGAGCGGAAAGATGTTGTTCTACATCCAGAACAACCTCTGCTAAAATTCCTGAAAAATTTTGCAAAAAGTTGTTGACTTTATCTACAAGGTGTGGTATAATAATCTTAACAACAGC AGGACGC (SEQ IDNO:2), and GAGGAATCATAGAATTTTGTCAAAATAATTTTATTGACAACGTCTTAITAACGTTGATATAATITAAATTFATTGACAAAAATGGGCTCGTGTTGTACAATAAATGTAGTGAGGTGGATGCAATG (SEQ ID NO:3). Additional preferred promotersinclude tef (the translational elongation factor (TEF) promoter) and pyc(the pyruvate carboxylase (PYC) promoter), which promote high levelexpression in Bacillus (e.g., Bacillus subtilis). Additional preferredpromoters, for example, for use in Gram positive microorganisms include,but are not limited to, amy and SPO2 promoters. Additional preferredpromoters, for example, for use in Gram negative microorganisms include,but are not limited to, cos, tac, trp, tet, typ-tet, lpp, lac, lpp-lac,lacIQ, T7, T5, T3, gal, trc, ara, SP6, λ-PR or λ-PL.

In another embodiment, a recombinant nucleic acid molecule of thepresent invention includes a terminator sequence or terminator sequences(e.g., transcription terminator sequences). The term “terminatorsequences” includes regulatory sequences that serve to terminatetranscription of mRNA. Terminator sequences (or tandem transcriptionterminators) can further serve to stabilize mRNA (e.g., by addingstructure to mRNA), for example, against nucleases.

In yet another embodiment, a recombinant nucleic acid molecule of thepresent invention includes sequences that allow for detection of thevector containing said sequences (i.e., detectable and/or selectablemarkers), for example, genes that encode antibiotic resistance sequencesor that overcome auxotrophic mutations, for example, trpC, drug markers,fluorescent markers, and/or colorimetric markers (e.g.,lacZ/β-galactosidase). In yet another embodiment, a recombinant nucleicacid molecule of the present invention includes an artificial ribosomebinding site (RBS) or a sequence that gets transcribed into anartificial RBS. The term “artificial ribosome binding site (RBS)”includes a site within an mRNA molecule (e.g., coded with in DNA) towhich a ribosome binds (e.g., to initiate translation) which differsfrom a native RBS (e.g., a RBS found in a naturally-occurring gene) byat least one nucleotide. Preferred artificial RBSs include about 5-6,7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26,27-28, 29-30 or more nucleotides of which about 1-2, 3-4, 5-6, 7-8,9-10, 11-12, 13-15 or more differ from the native RBS (e.g., the nativeRBS of a gene of interest, for example, the native panB RBS.TAAACATGAGGAGGAGAAAACATG (SEQ ID NO:4) or the native panD RBSATTCGAGAAATGGAGAGAATATAATATG (SEQ ID NO:5)). Preferably, nucleotidesthat differ are substituted such that they are identical to one or morenucleotides of an ideal RBS when optimally aligned for comparisons.Ideal RBSs include, but are not limited to, AGAAAGGAGGTGA (SEQ ID NO:6),TTAAGAAAGGAGGTGANNNNATG (SEQ ID NO: 7), TTAGAAAGGAGGTGANNNNNATG (SEQ IDNO: 8), AGAAAGGAGGTGANNNNNNNATG (SEQ ID NO: 9), andAGAAAGGAGGTGANNNNNNATG (SEQ ID NO: 10). Artificial RBSs can be used toreplace the naturally-occurring or native RBSs associated with aparticular gene. Artificial RBSs preferably increase translation of aparticular gene. Preferred artificial RBSs (e.g., RBSs for increasingthe translation of panB, for example, of B. subtilis panB) includeCCCTCTAGAAGGAGGAGAAAACATG (SEQ ID NO:11) and CCCTCTAGAGGAGGAGAAAACATG(SEQ ID NO:12). Preferred artificial RBSs (e.g., RBSs for increasing thetranslation of panD, for example, of B. subtilis panD) includeTTAGAAAGGAGGATTTAAATATG (SEQ ID NO:13), TTAGAAAGGAGGTTTAATTAATG (SEQ IDNO:14), TTAGAAAGGAGGTGATITAAATG (SEQ ID NO:15), TTAGAAAGGAGGTGTTTAAAATG(SEQ ID NO: 16), ATTCGAGAAAGGAGG TGAATATAATATG (SEQ ID NO:17),ATTCGAGAAAGGAGGTGAATAATAATG (SEQ ID NO: 18), andATTCGTAGAAAGGAGGTGAATTAATATG (SEQ ID NO:19).

The present invention further features vectors (e.g., recombinantvectors) that include nucleic acid molecules (e.g., genes or recombinantnucleic acid molecules comprising said genes) as described herein. Theterm “recombinant vector” includes a vector (e.g., plasmid, phage,phasmid, virus, cosmid or other purified nucleic acid vector) that hasbeen altered, modified or engineered such that it contains greater,fewer or different nucleic acid sequences than those included in thenative or natural nucleic acid molecule from which the recombinantvector was derived. Preferably, the recombinant vector includes abiosynthetic enzyme-encoding gene or recombinant nucleic acid moleculeincluding said gene, operably linked to regulatory sequences, forexample, promoter sequences, terminator sequences and/or artificialribosome binding sites (RBSs), as defined herein. In another embodiment,a recombinant vector of the present invention includes sequences thatenhance replication in bacteria (e.g., replication-enhancing sequences).In one embodiment, replication-enhancing sequences function in E. coli.In another embodiment, replication-enhancing sequences are derived frompBR322.

In yet another embodiment, a recombinant vector of the present inventionincludes antibiotic resistance sequences. The term “antibioticresistance sequences” includes sequences which promote or conferresistance to antibiotics on the host organism (e.g., Bacillus). In oneembodiment, the antibiotic resistance sequences are selected from thegroup consisting of cat (chloramphenicol resistance) sequences, tet(tetracycline resistance) sequences, erm (erythromycin resistance)sequences, neo (neomycin resistance) sequences, kan (kanamycinresistence) sequences and spec (spectinomycin resistance) sequences.Recombinant-vectors of the present invention can further includehomologous recombination sequences (e.g., sequences designed to allowrecombination of the gene of interest into the chromosome of the hostorganism). For example, bpr, vpr, or amyE sequences can be used ashomology targets for recombination into the host chromosome. It willfurther be appreciated by one of skill in the art that the design of avector can be tailored depending on such factors as the choice ofmicroorganism to be genetically engineered, the level of expression ofgene product desired and the like.

III. Recombinant Microorganisms

The present invention further features microorganisms, i.e., recombinantmicroorganisms, that include vectors or genes (e.g., wild-type and/ormutated genes) as described herein. As used herein, the term“recombinant microorganism” includes a microorganism (e.g., bacteria,yeast cell fungal cell, etc.) that has been genetically altered,modified or engineered (e.g., genetically engineered) such that itexhibits an altered, modified or different genotype and/or phenotype(e.g., when the genetic modification affects coding nucleic acidsequences of the microorganism) as compared to the naturally-occurringmicroorganism from which it was derived.

In one embodiment, a recombinant microorganism of the present inventionis a Gram positive organism (e.g., a microorganism which retains basicdye, for example, crystal violet, due to the presence of a Gram-positivewall surrounding the microorganism). In a preferred embodiment, therecombinant microorganism is a microorganism belonging to a genusselected from the group consisting of Bacillus. Cornyebacterium (e.g.,Cornyebacterium glutamicum), Lactobacillus, Lactococci and Streptomyces.In a more preferred embodiment, the recombinant microorganism is of thegenus Bacillus. In another preferred embodiment, the recombinantmicroorganism is selected from the group consisting of Bacillussubtilis, Bacillus lenitimorbus, Bacillus lentus, Bacillus firmus,Bacillus pantothenticus, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillusmegaterium, Bacillus pumilus, Bacillus thuringiensis, Bacillushalodurans, and other Group 1 Bacillus species, for example, ascharacterized by 16S rRNA type. In another preferred embodiment, therecombinant microorganism is Bacillus brevis or Bacillusstearothermophilus. In another preferred embodiment, the recombinantmicroorganism is selected from the group consisting of Bacilluslicheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, andBacillus pumilus.

In another embodiment, the recombinant microorganism is a Gram negative(excludes basic dye) organism. In a preferred embodiment, therecombinant microorganism is a microorganism belonging to a genusselected from the group consisting of Salmonella(e.g., Salmonellatyphimurium), Escherichia, Klebsiella, Serratia, and Proteus. In a morepreferred embodiment, the recombinant microorganism is of the genusEscherichia. In an even more preferred embodiment, the recombinantmicroorganism is Escherichia coli. In another embodiment, therecombinant microorganism is Saccharomyces (e.g., Saccharomycescerevisiae).

A preferred “recombinant” microorganism of the present invention is amicroorganism having a deregulated pantothenate biosynthesis pathway orenzyme, a deregulated isoleucine valine (ilv) biosynthetic pathway orenzyme and/or a modified or deregulated methylenetetrahydrofolate (MTF)biosynthetic pathway or enzyme and/or a modified or term “deregulated”or “deregulation” includes the alteration or modification of at leastone gene in a microorganism that encodes an enzyme in a biosyntheticpathway, such that the level or activity of the biosynthetic enzyme inthe microorganism is altered or modified. Preferably, at least one genethat encodes an enzyme in a biosynthetic pathway is altered or modifiedsuch that the gene product is enhanced or increased. The phrase“deregulated pathway” includes a biosynthetic pathway in which more thanone gene that encodes an enzyme in a biosynthetic pathway is altered ormodified such that the level or activity of more than one biosyntheticenzyme is altered or modified. The ability to “deregulate” a pathway(e.g., to simultaneously deregulate more than one gene in a givenbiosynthetic pathway) in a microorganism in some cases arises from theparticular phenomenon of microorganisms in which more than, one enzyme(e.g., two or three biosynthetic enzymes) are encoded by genes occurringadjacent to one another on a contiguous piece of genetic material termedan “operon” (defined herein). Due to the coordinated regulation of genesincluded in an operon, alteration or modification of the single promoterand/or regulatory element can result in alteration or modification ofthe expression of each gene product encoded by the operon. Alteration ormodification of a regulatory element can include, but is not limited toremoving the endogenous promoter and/or regulatory element(s), addingstrong promoters, inducible promoters or multiple promoters or removingregulatory sequences such that expression of the gene products ismodified, modifying the chromosomal location of a gene or operon,altering nucleic acid sequences adjacent to a gene or operon (or withinan operon) such as a ribosome binding site, increasing the copy numberof a gene or operon, modifying proteins (e.g., regulatory proteins,suppressors, enhancers, transcriptional activators and the like)involved in transcription of a gene or operon and/or translation of agene product or gene products of a gene or operon, respectively, or anyother conventional means of deregulating expression of genes routine inthe art (including but not limited to use of antisense nucleic acidmolecules, for example, to block expression of repressor proteins).Deregulation can also involve altering the coding region of one or moregenes to yield, for example, an enzyme that is feedback resistant or hasa higher or lower specific activity.

In another preferred embodiment, a recombinant microorganism is designedor engineered such that at least one pantothenate biosynthetic enzyme,at least one isoleucine-valine biosynthetic enzyme, and/or at least oneMTF biosynthetic enzyme is overexpressed. The term “overexpressed” or“overexpression” includes expression of a gene product (e.g., abiosynthetic enzyme) at a level greater than that expressed prior tomanipulation of the microorganism or in a comparable microorganism whichhas not been manipulated. In one embodiment the microorganism can begenetically designed or engineered to overexpress a level of geneproduct greater than that expressed in a comparable microorganism whichhas not been engineered.

Genetic, engineering can include, but is not limited to, altering ormodifying regulatory sequences or sites associated with expression of aparticular gene (e.g., by adding strong promoters, inducible promotersor multiple promoters or by removing regulatory sequences such thatexpression is constitutive), modifying the chromosomal location of aparticular gene, altering nucleic acid sequences adjacent to aparticular gene such as a ribosome binding site, increasing the copynumber of a particular gene, modifying proteins (e.g., regulatoryproteins, suppressors, enhancers, transcriptional activators and thelike) involved in transcription of a particular gene and/or translationof a particular gene product, or any other conventional means ofderegulating expression of a particular gene routine in the art(including but not limited to use of antisense nucleic acid molecules,for example, to block expression of repressor proteins). Geneticengineering can also include deletion of a gene, for example, to block apathway or to remove a repressor.

In another embodiment, the microorganism can be physically orenvironmentally manipulated to overexpress a level of gene productgreater than that expressed prior to manipulation of the microorganismor in a comparable microorganism which has not been manipulated. Forexample, a microorganism can be treated with or cultured in the presenceof an agent known or suspected to increase transcription of a particulargene and/or translation of a particular gene product such thattranscription and/or translation are enhanced or increased.Alternatively, a microorganism can be cultured at a temperature selectedto increase transcription of a particular gene and/or translation of aparticular gene product such that transcription and/or translation areenhanced or increased.

IV. Culturing and Fermenting Recombinant Microorganisms

The term “culturing” includes maintaining and/or growing a livingmicroorganism of the present invention (e.g., maintaining and/or growinga culture or strain). In one embodiment, a microorganism of theinvention is cultured in liquid media. In another embodiment, amicroorganism of the invention is cultured in solid media or semi-solidmedia. In a preferred embodiment, a microorganism of the invention iscultured in media (e.g., a sterile, liquid medium) comprising nutrientsessential or beneficial to the maintenance and/or growth of themicroorganism (e.g., carbon sources or carbon substrate, for examplecarbohydrate, hydrocarbons, oils, fats, fatty acids, organic acids, andalcohols, nitrogen sources, for example, peptone, yeast extracts, meatextracts, malt extracts, soy meal, soy flour, soy grits, urea, ammoniumsulfate, ammonium, chloride, ammonium nitrate and ammonium phosphate;phosphorus sources, for example, phosphoric acid, sodium and potassiumsalts thereof; trace elements, for example, magnesium, iron, manganese,calcium, copper, zinc, boron, molybdenum, and/or cobalt salts; as wellas growth factors such as amino acids, vitamins, growth promoters andthe like).

Preferably, microorganisms of the present invention are cultured undercontrolled pH. The term “controlled pH” includes any pH which results inproduction of the desired product (e.g., pantoate and/or pantothenate).In one embodiment microorganisms are cultured at a pH of about 7. Inanother embodiment, microorganisms are cultured at a pH of between 6.0and 8.5. The desired pH may be maintained by any number of methods knownto those skilled in the art.

Also preferably, microorganisms of the present invention are culturedunder controlled aeration. The term “controlled aeration” includessufficient aeration (e.g., oxygen) to result in production of thedesired product (e.g., pantoate and/or pantothenate). In one embodiment,aeration is controlled by regulating oxygen levels in the culture, forexample, by regulating the amount of oxygen dissolved in culture media.Preferably, aeration of the culture is controlled by agitating theculture. Agitation may be provided by a propeller or similar mechanicalagitation equipment, by revolving or shaking the cuture vessel (e.g.,tube or flask) or by various pumping equipment. Aeration may be furthercontrolled by the passage of sterile air or oxygen through the medium(e.g., through the fermentation mixture). Also preferably,microorganisms of the present invention are cultured without excessfoaming (e.g., via addition of antifoaming agents).

Moreover, microorganisms of the present invention can be cultured undercontrolled temperatures. The term “controlled temperature” includes anytemperature which results in production of the desired product (e.g.,pantoate and/or pantothenate). In one embodiment, controlledtemperatures include temperatures between 15° C. and 95° C. In anotherembodiment, controlled temperatures include temperatures between 15° C.and 70° C. Preferred temperatures are between 20° C. and 55° C., morepreferably between 30° C. and 50° C.

Microorganisms can be cultured (e.g., maintained and/or grown) in liquidmedia and preferably are cultured, either continuously orintermittently, by conventional culturing methods such as standingculture, test tube culture, shaking culture (e.g., rotary shakingculture, shake flask culture, etc.), aeration spinner culture orfermentation. In a preferred embodiment, the microorganisms are culturedin shake flasks. In a more preferred embodiment, the microorganisms arecultured in a fermentor (e.g., a fermentation process). Fermentationprocesses of the present invention include, but are not limited to,batch, fed-batch and continuous processes or methods of fermentation.The phrase “batch process” or “batch fermentation” refers to a system inwhich the composition of media, nutrients, supplemental additives andthe like is set at the beginning of the fermentation and not subject toalteration during the fermentation, however, attempts may be made tocontrol such factors as pH and oxygen concentration to prevent excessmedia acidification and/or microorganism death. The phrase “fed-batchprocess” or “fed-batch” fermentation refers to a batch fermentation withthe exception that one or more substrates or supplements are added(e.g., added in increments or continuously) as the fermentationprogresses. The phrase “continuous process” or “continuous fermentation”refers to a system in which a defined fermentation media is addedcontinuously to a fermentor and an equal amount of used or “conditioned”media is simultaneously removed, preferably for recovery of the desiredproduct (e.g., pantoate and/or pantothenate). A variety of suchprocesses have been developed and are well-known in the art.

The phrase “culturing under conditions such that a desired compound isproduced” includes maintaining and/or growing microorganisms underconditions (e.g., temperature, pressure, pH, duration, etc.) appropriateor sufficient to obtain production of the desired compound or to obtaindesired yields of the particular compound being produced. For example,culturing is continued for a time sufficient to produce the desiredamount of a compound (e.g., pantoate and/or pantothenate). Preferably,culturing is continued for a time sufficient to substantially reachsuitable production of the compound (e.g., a time sufficient to reach asuitable concentration of pantoate and/or pantothenate or suitable ratioof pantoate and/or pantothenate:HMBPA). In one embodiment, culturing iscontinued for about 12 to 24 hours. In another embodiment, culturing iscontinued for about 24 to 36-hours, 36 to 48 hours, 48 to 72 hours, 72to 96 hours, 96 to 120 hours, 120 to 144 hours, or greater than 144hours. In yet another embodiment, microorganisms are cultured underconditions such that at least about 5 to 10 g/L of compound are producedin about 36 hours, at least about 10 to 20 g/L compound are produced inabout 48 hours, or at least about 20 to 30 g/L compound in about 72hours. In yet another embodiment, microorganisms are cultured underconditions such that at least about 5 to 20 g/L of compound are producedin about 36 hours, at least about 20 to 30 g/L compound are produced inabout 48 hours, or at least about 30 to 50 or 60 g/L compound in about72 hours. In yet another embodiment, microorganisms are cultured underconditions such that at least about 40 to 60 g/L of compound areproduced in about 36 hours, or at least about 60 to 90 g/L compound areproduced in about 48 hours. It will be appreciated by the skilledartisan that values above the upper limits of the ranges recited may beobtainable by the processes described herein, for example, in aparticular fermentation run or with a particular engineered strain.

Preferably, a production method of the present invention results inproduction of a level of pantothenate that is “enhanced as compared toan appropriate control”. The term “appropriate control”, as definedherein, includes any control recognized by the skilled artisan as beingapproriate for determining enhanced, increased, or elevated levels ofdesired product. For example, where the process features culturing amicroorganism having a deregulated pantothenate biosynthetic pathway andsaid microorganism further has a deregulated MTF biosynthetic pathway(e.g., has been engineered such that at least one MTF biosyntheticenzyme is deregulated, for example, overexpressed) an appropriatecontrol includes a culture of the microorganism before or absentmanipulation of the MTF enzyme or pathway (i.e., having only thepantothenate biosynthetic pathway deregulated). Likewise, where theprocess features culturing a microorganism having a deregulatedpantothenate biosynthetic pathway and a deregulated ilv biosyntheticpathway and said microorganism further has a deregulated MTFbiosynthetic pathway (i.e., has been engineered such that at least oneMTF biosynthetic enzyme is deregulated, for example, overexpressed) anappropriate control includes a culture of the microorganism before orabsent manipulation of the MTF enzyme or pathway (i.e., having only thepantothenate biosynthetic pathway and ilv biosynthetic pathwayderegulated). Comparison need not be performed in each process practicedaccording to the present invention. For example, a skilled artisan candetermine appropriate controls empirically from performing a series ofreactions (e.g., test tube cultures; shake flask cultures,fermentations), for example, under the same or similar conditions.Having appreciated a routine production level, for example, by aparticular strain, the artisan is able to recognize levels that areenhanced, increased or elevated over such levels. In other words,comparison to an appropriate control includes comparison to apredetermined values (e.g., a predetermined control).

Thus, in an embodiment wherein an appropriately engineered strainproduces 40 g/L pantothenate in 36 hours (prior to manipulation suchthat pantothenate production is enhanced), production of 50, 60, 70 ormore g/L pantothenate (after manipulation, for example, manipulationsuch that at least one MTF biosynthetic enzyme is overexpressed)exemplifies enhanced production. Likewise, in an embodiment wherein anappropriately engineered strain produces 50 g/L pantothenate in 48 hours(prior to manipulation such that pantothenate production is enhanced),production of 60, 70, 80, 90 or more g/L pantothenate (aftermanipulation, for example, manipulation such that at least one MTFbiosynthetic enzyme is overexpressed) exemplifies enhanced production.

The methodology of the present invention can further include a step ofrecovering a desired compound (e.g., pantoate and/or pantothenate). Theterm “recovering” a desired compound includes extracting, harvesting,isolating or purifying the compound from culture media. Recovering thecompound can be performed according to any conventional isolation orpurification methodology known in the art including, but not limited to,treatment with a conventional resin (e.g., anion or cation exchangeresin, non-ionic adsorption resin, etc.), treatment with a conventionaladsorbent (e.g., activated charcoal, silicic acid, silica gel,cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g.,with a conventional solvent such as an alcohol, ethyl acetate, hexaneand the like), dialysis, filtration, concentration, crystallization,recrystallization, pH adjustment, lyophilization and the like. Forexample, a compound can be recovered from culture media by firstremoving the microorganisms from the culture. Media are then passedthrough or over a cation exchange resin to remove cations and thenthrough or over an anion exchange resin to remove inorganic anions andorganic acids having stronger acidities than the compound of interest.The resulting compound can subsequently be converted to a salt (e.g., acalcium salt) as described herein.

Preferably, a desired compound of the present invention is “extracted”,“isolated” or “purified” such that the resulting preparation issubstantially free of other media components (e.g., free of mediacomponents and/or fermentation byproducts). The language “substantiallyfree of other media components” includes preparations of the desiredcompound in which the compound is separated from media components orfermentation byproducts of the culture from which it is produced. In oneembodiment, the preparation has greater than about 80% (by dry weight)of the desired compound (e.g., less than about 20% of other mediacomponents or fermentation byproducts), more preferably greater thanabout 90% of the desired compound (e.g., less than about 10% of othermedia components or fermentation byproducts), still more preferablygreater than about 95% of the desired compound (e.g., less than about 5%of other media components or fermentation byproducts), and mostpreferably greater than about 98-99% desired compound (e.g., less thanabout 1-2% other media components or fermentation byproducts). When thedesired compound has been derivatized to a salt, the compound ispreferably further free of chemical contaminants associated with theformation of the salt. When the desired compound has been derivatized toan alcohol, the compound is preferably further free of chemicalcontaminants associated with the formation of the alcohol.

In an alternative embodiment, the desired compound is not purified fromthe microorganism, for example, when the microorganism is biologicallynon-hazardous (e.g., safe). For example, the entire culture (or culturesupernatant) can be used as a source of product (e.g., crude product).In one embodiment, the culture (or culture supernatant) is used withoutmodification. In another embodiment, the culture (or culturesupernatant) is concentrated. In yet another embodiment, the culture (orculture supernatant) is dried or lyophilized.

In yet another embodiment, the desired compound is partially purified.The term “partially purified” includes media preparations that have hadat least some processing, for example, treatment (e.g., batch treatment)with a commercial resin. In preferred embodiments, the “partiallypurified” preparation has greater than about 30% (by dry weight) of thedesired compound, preferably greater than about 40% of the desiredcompound, more preferably greater than about 50% of the desiredcompound, still more preferably greater than about 60% of the desiredcompound, and most preferably greater than about 70% desired compound.“Partially purified” preparations also preferably have 80% or less (bydry weight) of the desired compound (i.e., are less pure than“extracted”, “isolated” or “purified” preparations, as defined herein).

Depending on the biosynthetic enzyme or combination of biosyntheticenzymes manipulated, it may be desirable or necessary to provide (e.g.,feed) microorganisms of the present invention at least one biosyntheticprecursor such that the desired compound or compounds are produced. Theterm “biosynthetic precursor” or “precursor” includes an agent orcompound which, when provided to, brought into contact with, or includedin the culture medium of a microorganism, serves to enhance or increasebiosynthesis of the desired product. In one embodiment, the biosyntheticprecursor or precursor is aspartate. In another embodiment, thebiosynthetic precursor or precursor is β-alanine. The amount ofaspartate or β-alanine added is preferably an amount that results in aconcentration in the culture medium sufficient to enhance productivityof the microorganism (e.g., a concentration sufficient to enhanceproduction of pantoate and/or pantothenate). Biosynthetic precursors ofthe present invention can be added in the form of a concentratedsolution or suspension (e.g., in a suitable solvent such as water orbuffer) or in the form of a solid (e.g., in the form of a powder).Moreover, biosynthetic precursors of the present invention can be addedas a single aliquot, continuously or intermittently over a given periodof time. The term “excess β-alanine” includes β-alanine levels increasedor higher that those routinely utilized for culturing the microorganismin question. For example, culturing the Bacillus microorganismsdescribed in the instant Examples is routinely done in the presence ofabout 0-0.01 g/L β-alanine. Accordingly, excess β-alanine levels caninclude levels of about 0.01-1, preferably about 1-20 g/L.

In yet another embodiment, the biosynthetic precursor is valine. In yetanother embodiment, the biosynthetic precursor is α-ketoisovalerate.Preferably, valine or α-ketoisovalerate is added in an amount thatresults in a concentration in the medium sufficient for production ofthe desired product (e.g., pantoate and/or pantothenate) to occur. Theterm “excess α-KIV” includes α-KIV levels increased or higher that thoseroutinely utilized for culturing the microorganism in question. Forexample, culturing the Bacillus microorganisms described in the instantExamples is routinely done in the presence of about 0-0.01 g/L α-KIV.Accordingly, excess α-KIV levels can include levels of about 0.01-1preferably about 1-20 g/L α-KIV. The term “excess valine” includesvaline levels increased or higher that those routinely utilized forculturing the microorganism in question. For example, culturing theBacillus microorganisms described in the instant Examples is routinelydone in the presence of about 0-0.5 g/L valine. Accordingly, excessvaline levels can include levels of about 0.5-5 g/L, preferably about5-20 g/L valine.

In yet another embodiment, the biosynthetic precursor is serine.Preferably, serine is added in an amount that results in a concentrationin the medium sufficient for production of the desired product (e.g.,pantoate and/or pantothenate) to occur. Excess serine (as definedherein) can also be added according to the production processesdescribed herein, for example, for the enhanced production ofpantothenate. The skilled artisan will appreciate that extreme excessesof biosynthetic precursors can result in microorganism toxicity.Biosynthetic precursors are also referred to herein as “supplementalbiosynthetic substrates”.

Another aspect of the present invention includes biotransformationprocesses which feature the recombinant microorganisms described herein.The term “biotransformation process”, also referred to herein as“bioconversion processes”, includes biological processes which resultsin the production (e.g., transformation or conversion) of appropriatesubstrates and/or intermediate compounds into a desired product.

The microorganism(s) and/or enzymes used in the biotransformationreactions are in a form allowing them to perform their intended function(e.g., producing a desired compound). The microorganisms can be wholecells, or can be only those portions of the cells necessary to obtainthe desired end result. The microorganisms can be suspended (e.g., in anappropriate solution such as buffered solutions or media), rinsed (e.g.,rinsed free of media from culturing the microorganism), acetone-dried,immobilized (e.g., with polyacrylamide gel or k-carrageenan or onsynthetic supports, for example, beads, matrices and the like), fixed,cross-linked or permeablized (e.g., have permeablized membranes and/orwalls such that compounds, for example, substrates, intermediates orproducts can more easily pass through said membrane or wall).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example I Panto-Compound Production Strains

In developing Bacillus strains for the production of pantothenate,various genetic manipulations are made to genes and enzymes involved inthe pantothenate biosynthetic pathway and the isoleucine-valine (ilv)pathway (FIG. 1) as described in U.S. patent application Ser. No.09/400,494 and U.S. patent application Ser. No. 09/667,569. For example,strains having a deregulated panBCD operon and/or having deregulatedpanE1 exhibit enhanced pantothenate production (when cultured in thepresence of β-alanine and α-ketoisovalerate (α-KIV)). Strains furtherderegulated for ilvBNC and ilvD exhibit enhanced pantothenate productionin the presence of only β-alanine. Moreover, it is possible to achieveβ-alanine independence by further deregulating panD.

An exemplary pantothenate production strain is PA824, a tryptophanprototroph, Spec and Tet resistant, deregulated for panBCD at the panBCDlocus, deregulated for panE1 at the panE1 locus (two genes in the B.subtilis genome are homologous to E. coli panE, panE1 and panE2, theformer encoding the major ketopantoate reductase involved inpantothenate production, while panE2 does not contribute to pantothenatesynthesis (U.S. patent application Ser. No. 09/400,494), deregulated forilvD at the ilvD locus, overexpressing an ilvBNC cassette at the amyElocus, and overexpressing panD at the bpr locus. PA824 routinely yieldsapproximately 40-50 g/L pantothenate, when cultured for 48 hours in 14 Lfermentor vessels according to standard fermentation procedures (seee.g., provisional Patent Application Ser. No. 60/263,053 or provisionalPatent Application Ser. No. 60/262,995, incorporated by referenceherein). Briefly, batch media (4.5 L) containing trace elements isinoculated with shake flask cultures of PA824. The fermentations arecontrolled for temperature (e.g., 43° C.), dissolved O₂, and pH, and arerun as a glucose limited fed batch process. After the initial batchedglucose is consumed, glucose concentrations are maintained between about0 and 1 g/L by continuous feeding of fresh FEED media pH is set at 7.2,monitored, and maintained by feeding either a NH₃- or a H₃PO₄-solution.The dissolved oxygen concentration. [pO₂] is maintained at about 10-30%by regulation of the agitation and aeration rate. Foaming is controlledby addition of an appropriate antifoam agent. The pantothenate titer inthe fermentation broth is determined (by HPLC analysis) after removal ofthe cells by centrifugation.

A second exemplary strain is PA668. PA668 is a derivative of PA824 thatcontains extra copies of P₂₆panB amplified at the vpr and/or panB locus.PA668 was constructed using a panB expression vector (pAN636) whichallows for selection of multiple copies using chloramphenicol. Briefly,a pAN636 NotI restriction fragment (excluding vector sequences) wasligated and then used to transform PA824 with selection on platescontaining 5 μg/ml chloramphenicol. Transformants resistant to 30 μg/mlchloramphenicol were isolated and screened for pantothenate productionin 48 hour test tube cultures. The isolates produce about 10 percentmore pantothenate than PA824. In 10-L fermentations, a first strain,PA668-24, produces pantothenate in amounts comparable to PA824 culturedunder similar conditions (e.g., ˜45-50 g/L at 36 hours). After 36 hours,when pantothenate production routinely begins to slow with PA824,PA668-2A continues to produce significant levels of pantothenate (e.g.,˜60-65 g/l pantothenate at 48 hours). A second strain, PA668-24,produces pantothenate at an even faster rate, reaching 60-70 g/L after48 hours.

A third production strain, PA721B-39, was engineered to further includean amplifiable P₂₆panBpanD cassette as follows. First, a singleexpression cassette was constructed that is capable of integrating bothpanB and panD at the bpr locus. Combining both genes into one expressioncassette simplifies the resulting strain by eliminating an antibioticresistance marker. The P₂₆panBpanD expression cassette was constructedto include each of two different panD ribosome binding sites (the RBSshaving previously been synthesized and tested in International Public.No. WO 01/21772 and U.S. Patent Application Ser. No. 60/262,995). Thecassette further included the synthetic panB gene ribosome binding site(RBS1), but the design permits future alteration of the panB RBS bysimple oligonucleotide cassette substitution. In the first step ofconstruction, the panB gene was joined to the two panD gene cassettes asillustrated in FIG. 3 for the construction of pAN665. Next, theresulting panBpanD cassettes were transferred to B. subtilis expressionvector pOTP61 as illustrated in FIG. 4. A summary of the essentialfeatures of each plasmid (pAN670 and pAN674) constructed is presented inTable 1.

TABLE 1 Plasmids containing various B. subtilis panBpanD gene expressioncassettes. Plasmid panD RBS Vector Host strain pAN665 Standard pASK- E.coli 1BA3 pAN670 ″ pOTP61 B. subtilis pAN669 ND-C2 pASK- E. coli 1BA3pAN674 ″ pOTP61 B. subtilis

These new plasmids combine production of extra PanB and PanD from asingle vector and were predicted to produce increased levels of PanBrelative to the panB expression vector (pAN636) present in PA668. Thestrategy to install the P26 panBpanD vectors in pantothenate productionstrains took advantage of genetic linkage between bpr and panE1. Aderivative of PA824 was first constructed that is cured of the residentpanD expression cassette by transforming the strain with chromosomal DNAisolated from PA930 (panE1::cat) and selecting for resistance tochloramphenicol. The resulting transformants were screened forsensitivity to tetracycline, and two Tet-sensitive isolates named PA715were saved. This strain is the host strain for testing the P26panBpanDvectors (see below). In order to restore the P26panE1 cassette in PA715,each vector was first transformed into a strain (PA328) that containsP26panE1 but does not contain a cassette integrated at the bpr locus.PA328 does contain the P26 panBCD locus although it is not engineeredfor overproduction of α-KIV. Transformants of PA328 resistant totetracycline were obtained using the appropriate NotI restrictionfragments from the two vectors and the resulting strains were namedPA710 and PA714.

The next step was to transfer the cassettes into PA715 so they could beevaluated in the PA824 strain background. This was accomplished byisolating chromosomal DNA from strains PA710 and PA714 and using each ofthe two DNAs separately to transform PA715, with selection forresistance to tetracycline. Tetracycline resistant transformants werescreened for sensitivity to chloramphenicol; this identifies the desiredtransformants that have also acquired the P26panE1 gene from the donorDNA by linkage with the P26panBpanD cassettes at the bpr locus.Chloramphenicol-sensitive isolates derived from transformations in whichPA710 or PA714 chromosomal DNA was used as the donor were obtained. Theisolates that produced the highest pantothenate titers in test tubeculture assays were saved. These strains were named PA717 and PA721,respectively. Duplicate test tube cultures of the new strains, as wellas PA824 and PA715, were grown in SVY+10 g/L aspartate at 43° C. for 48hours and then assayed for pantothenate, HMBPA, and β-alanine. Inaddition, extracts from each of the strains were run on a SDS-PAGE gel.The results of the test tube culture assays are presented in Table 2.

TABLE 2 Production of pantothenate by strains PA717 and PA721 grown inSVY plus 10 g/l aspartate. panBD [pan] [HMBPA] [β-ala] Strain cassette(g/L) (g/L) (g/L) PA824 — 4.9 0.94 2.5 ″ 4.6 0.79 2.3 PA715 NONE 1.7<0.1 0.5 ″ ″ 1.7 <0.1 0.4 PA717-24 pAN670 4.8 0.34 1.3 ″ ″ 4.9 0.40 1.3PA721-35 pAN674 5.7 0.50 1.4 ″ ″ 5.3 0.40 1.3 PA721-39 pAN674 4.1 0.382.0 ″ ″ 4.6 0.40 2.2

As expected, each of the new strains produced more pantothenate andβ-alamine than PA715. Two of the strain (PA717-24 and PA721-39) producedabout as much pantothenate as PA824 while PA721-35 produced morepantothenate PA824. All tree of the new strains produced less HMBPA thanPA824. The protein gel analysis showed that the three new strainsproduce more PanB than any of the control strains.

Strains PA717-24, PA721-35, and PA721-39 were also evaluated in shakeflask cultures in a soy-flour based medium. As shown Table 3, thesestrains with the amplifiable P₂₆panBpanD cassette produced pantothenateand HMBPA at levels similar to the levels seen with PA668-2 and PA668-24which both contain separate amplifiable P₂₆panB and P₂₆panD cassettes.

TABLE 3 Shake Flask Experiment 48 Hours HMBPA PAN Medium Strain (g/l)(g/l) Soy flour + Glucose PA668-2 1.2 6.8 PA668-24 1.6 5.2 PA717-24 2.05.9 PA721-35 2.6 7.0 PA721-39 2.5 8.6 Soy flour + Maltose PA668-2 0.09.0 PA668-24 0.4 10.4 PA717-24 0.7 8.6 PA721-35 1.0 9.2 PA721-39 0.4 9.1Conditions: 40 ml medium/200 ml baffled shake flask, 4X Bioshieldcovers, 300 rpm, 2.5% inoculum (1.0 ml). Soy Medium: 20 g/l Cargill200/20 soy flour, 8 g/l (NH4)2SO4, 5 g/l glutamate, 1 × PSTE, 0.1Mphosphate pH 7.2 and 0.3M MOPS pH 7.2. 60 g/l glucose or maltose w/10 mMMg and 1.4 mM Ca. Average of duplicate flasks.

In addition to producing pantothenate (as well as other panto-compoundsdepicted in FIG. 1 and described herein), it has been demonstrated thatcertain strains engineered for producing commercial quantities ofdesired panto-compound also produce a by-product identified as3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA)(also referredto herein as “β-alanine 2-(R)-hydroxyisolvalerate”, “β-alanine2-hydroxyisolvalerate”, “β-alanyl-α-hydroxyisovalarate” and/or“fantothenate”). (The term “fantothenate” is also abbreviated as “fan”herein.)

HMBPA is the condensation product of [R]-α-hydroxyisovaleric acid(α-HIV) and β-alanine, catalyzed by the PanC enzyme. α-HIV is generatedby reduction of α-KIV, a reaction that is catalyzed by the α-ketoreductases PanE (e.g., PanE1 and/or PanE2) and/or IlvC. Thus it has beenproposed that there exist at least two pathways in microorganisms thatcompete for α-KIV, the substrate for the biosynthetic enzyme PanB,namely the pantothenate biosynthetic pathway and the HMBPA biosyntheticpathway. (A third and fourth pathway competing for α-KIV are thoseresulting in the production of valine or leucine from α-KIV, see e.g.,FIG. 1). At least the pantothenate biosynthetic pathway and the HMBPAbiosynthetic pathway further produce competitive substrates for theenzyme PanC, namely α-HIV and pantoate. Production of HMBPA can havesignificant effects on pantothenate production. For example, the HMBPApathway can compete with the pantothenate pathway for precursors (α-KIVand β-alanine) and for some of the enzymes (PanC, PanD, PanE1, and/orIlvC). In addition, because the structure of HMBPA is similar to that ofpantothenate, it may have the undesirable property of negativelyregulating one or more steps in the pantothenate pathway. Based on theidentification of HMBPA, U.S. Provisional Patent Application Ser. No.60/262,995 teaches that production of pantothenate can be improved oroptimized by any means which favor use of substrates (α-KIV andβ-alanine) and/or enzymes (PanC, PanD, PanE1, and/or IlvC) inpantothenate biosynthetic processes as compared to HMBPA biosyntheticprocesses.

Example II Increasing Pantothenate Production by Increasing SerineAvailability

At least one method for optimizing pantothenate production involvesregulating the availability of serine in the microorganism cultures. Inparticular, it can be demonstrated that increasing the availability ofserine leads to increased pantothenate production (e.g., relative toHMBPA production), whereas decreasing the availability of serine leadsto decreased pantothenate production relative to HMBPA production. Thismethod is based on the understanding that the compound,methylenetetrahydrofolate (MTF), which is derived from serine, donates ahydroxymethyl group to α-KIV during the pantothenate biosyntheticreaction to yield ketopantoate (see e.g., FIGS. 1 and 2). Thus,regulating serine levels is one means of effectively regulatingketopantoate levels and, in turn, regulating pantoate and/orpantothenate production in appropriately engineered microorganisms. Todemonstrate this regulation, PA824 was grown in test tube cultures ofSVY glucose plus 5 g/L β-alanine and ±5 g/L serine for 48 hours and 43°C.

TABLE 4 Production of pantothenate and HMBPA by PA824 with and withoutthe addition of serine serine added at 5 g/L OD₆₀₀ [pan] g/L [HMBPA] g/L− 16.3 4.9 0.84 − 14.0 4.5 0.80 + 13.1 6.4 0.56 + 12.9 6.0 0.62

As demonstrated by the data presented in Table 4, addition of serineincreases the level of production of pantothenate (while converselydecreasing HMBPA production).

Example III Engineering Bacterial Cells with Increased Amounts of SerineHydroxylmethyl Transferase, the glyA Gene Product

As an alternative to feeding serine, another method of increasing serinelevels and/or serine utilization levels (and accordingly,methylenetetrahydrofolate levels) in order to regulate pantothenateproduction levels is to increase synthesis or the activity of3-phosphoglycerate dehydrogenase or of serine hydroxymethyl transferase(the serA and glyA gene products, respectively), thereby increasingserine and methylenetetrahydrofolate biosynthesis in appropriatelyengineered microorganisms.

Expression of the glyA gene was increased by transforming B. subtiliscells with an expression cassette containing the B. subtilis glyA genecloned downstream of a strong, constitutive promoter. To construct theexpression cassette the primers RY417 and RY418 depicted in Table 5 wereused to amplify the glyA gene by PCR from chromosomal DNA isolated fromB. subtilis PY79.

TABLE 5 Primers used in the amplification of B. subtilis glyA and serASEQ ID NO: 20 RY405 CCCTCTAGAGGAGGAGAAAACATGTTTCGAGTATTGGTC TCAGACAAAATGSEQ ID NO: 21 RY406 CCCGGATCCAATTATGGCAGATCAATGAGCTTCACAGAC ACAA SEQ IDNO: 22 RY417 GGATCTAGAGGAGGTGTAAACATGAAACATTTACCTGCG CAAGACGAA SEQ IDNO: 23 RY418 CGGGGATCCCCCATCAACAATTACACACTTCTATTGATT CTAC

RY417 contains the RBS2 synthetic ribosome binding site just downstreamfrom an XbaI site. The amplified DNA was then cut with XbaI and BamHIand cloned between the XbaI and BamHI sites in vector pAN004 (FIG. 5) toyield plasmid pAN396 (FIG. 6; SEQ ID NO:24). The pAN004 vector containsthe phage SP01 P₂₆ promoter immediately upstream of the XbaI cloningsite to drive expression of the cloned glyA gene. Just downstream of theexpression cassette, pAN396 contains a cat gene that functions in B.subtilis. To transform B. subtilis, the NotI DNA fragment containing theP₂₆glyA cassette and cat gene was isolated from pAN396, self-ligated,and transformed into competent cells of B. subtilis PY79. Severalchloramphenicol resistant transformants were selected and named PA1007and PA1008. Chromosomal DNA was isolated from each of these strains andused to transform competent cells of PA721B-39 and PA824 to yieldstrains PA1011 and PA1014, respectively. SDS polyacrylamide gelelectrophoresis of cell extracts of selected isolates of PA1011 andPA1014 confirmed that these strains contained increased amounts of theglyA gene product as compared to their parent strains PA721B-39(described in Example I) and PA824 (described in International Public.No. WO 01/21772). To test the effect of increasing glyA expression onpantothenate production, PA1011 and PA1014 were grown in test tubecultures of SVY glucose plus 5 g/L β-alanine at 43° C. for 48 hours. Asshown by the data presented in Table 6, PA 014 produced morepantothenate (4.5 g/L) than its parent strain PA824 (3.2 g/L).Similarly, PA1011 produced on average more pantothenate (4.35 g/L) thanits parent strain PA721B-39 (4.05 g/L).

TABLE 6 Production of pantothenate and HMBPA by PA1011 and PA1014compared to PA721B-39 and PA824. Pantothenate HMBPA Strain OD₆₀₀ g/L g/LPA1014 #1 14 4.5 0.27 PA1014 #2 15 4.5 0.31 PA824 16 3.1 0.31 PA824 153.3 0.28 PA1011 #1 17 4.5 0.24 PA1011 #2 12 4.2 0.27 PA721B-39 18 4.00.22 PA721B-39 16 4.1 0.25

Example IV Engineering Bacterial Cells with Increased Amounts of3-Phosphoglycerate Dehydrogenase, the serA Gene Product

The product of the sera gene, 3-phosphoglycerate dehydrogenase, is thefirst committed enzyme in the pathway to serine biosynthesis (see FIG.2). Since serine is one of the substrates for the synthesis of MTF, weengineered the overexpression of the sera gene to increase serine levelsin the cell. In a manner similar to that described above for the glyAgene in Example III, expression of the serA gene was increased bytransforming B. subtilis cells with an expression cassette containingthe B. subtilis sera gene cloned downstream of a strong, constitutivepromoter. To construct the expression cassette the primers RY405 andRY406 depicted in Table 5 were used to amplify the sera gene by PCR fromchromosomal DNA isolated from B. subtilis PY79. The amplified DNA wasthen cut with XbaI and BamHI and cloned between the XbaI and BamHI sitesin vector pAN004 (FIG. 5) to yield plasmid pAN393 (FIG. 7; SEQ IDNO:25). To transform B. subtilis, the NotI DNA fragment containing theP26 sera cassette and cat gene was isolated from pAN393, self-ligated,and transformed into competent cells of B. subtilis PY79. Severalchloramphenicol resistant transformants were selected and named PA1004and PA1005. Chromosomal DNA was isolated from each of these strains andused to transform competent cells of PA721B-39 and PA824 to yieldstrains PA1010 and PA1013, respectively. SDS polyacrylamide gelelectrophoresis of cell extracts of selected isolates of PA1010 andPA1013 confirmed that these strains contained increased amounts of thesera gene product as compared to their parent strains PA721B-39 andPA824.

To test the effect of increasing sera expression on pantothenateproduction, PA1010 and PA1013 were grown in test tube cultures of SVYglucose plus 5 g/L β-alanine at 43° C. for 48 hours. As shown by thedata presented in Table 7, PA1010 produced on average more pantothenate(4.7 g/L) than its parent strain PA721B-39 (4.1 g/L). Similarly, PA1013produced on average more pantothenate (4.1 g/L) than its parent strainPA824 (3.1 g/L).

TABLE 7 Production of pantothenate and HMBPA by PA1010 and PA1013compared to PA721B-39 and PA824. Pantothenate HMBPA Strain OD₆₀₀ g/L g/LPA1010 #3 16 4.8 0.23 PA1010 #5 15 4.5 0.26 PA1010 #6 22 4.7 0.24PA721B-39 18 4.0 0.22 PA721B-39 16 4.1 0.25 PA1013 #2 14 3.3 0.25 PA1013#4 14 4.2 0.28 PA1013 #5 16 5.5 0.37 PA1013 #8 13 3.6 0.24 PA824 17 3.00.27 PA824 16 3.1 0.29

Example V Shake Flask and Fermentor Experiments with Strains withIncreased Expression of serA and glyA

Based on performance in test tubes, two strains with an amplifiable serAcassette and two strains with an amplifiable glyA cassette wereselected, one each from two parents, PA824 and PA721B-39. The fourstrains were grown beside the parents in shake flasks (Table 8). In Soyflour MOPS Glucose (SMG) medium, all of the 4 strains produced morepantothenate than their parent strains. In Soy flour MOPS Maltose (SMM)medium one out of the four strains appeared superior to the parentstrain.

The sera overexpressing strain and the glyA overexpressing strain fromeach parent were run simultaneously in 10-liter Chemap bench fermentors.The glyA overexpressing strain derived from PA824, PA1014-3, that hadgiven the highest pantothenate titer in SMM, also performed the best infermentors (Table 9). Strain PA1014-3 produced 71 g/l pantothenate in 36hours in the culture supernatant and 86 g/l pantothenate in 48 hours inthe culture supernatant compared to the parent PA824 which produced 41g/l and 46 g/l pantothenate, respectively. The sera strain, PA1012-4,also produced significantly more pantothenate than the PA824 control inthe culture supernatant, 52 g/l and 60 g/l at 36 and 48 hours,respectively. These results clearly demonstrate the effectiveness ofincreasing both glyA and sera.

The sera overexpressing and glyA overexpressing derivatives of PA721B-39were clearly improved over their parent strain as well. Both producedabout 80 g/l pantothenate (82 g/l and 79 g/l, respectively) in theculture supernatants in 48 hours. The effect of the increased PanBlevels in the PA721B-39 derivatives versus the PA824 derivativesmanifests itself in the reduction of HMBPA. PA721B-39 and itsderivatives produce less HMBPA after 48 hours than PA824 or evenPA668-24. Increasing GlyA also appears to lower the flow of carbon toHMBPA.

TABLE 8 Shake flask evaluation of pantothenate production strainsoverexpressing ser A or gly A. Carbon Added HMBPA Pantothenate sourceStrain cassette (g/l) (g/l) Glucose PA824 3.5 4.0 PA1012-4 serA 3.0 4.6PA1014-3 gly A 2.5 4.7 PA721B-39 0.9 5.0 PA1010-6 serA 1.9 9.6 PA1011-2gly A 1.7 10.0 Maltose PA824 1.2 10.4 PA1012-4 serA 0.8 9.8 PA1014-3 glyA 1.1 16.1 PA721B-39 0.6 11.6 PA1010-6 serA 0.5 10.2 PA1011-2 gly A 010.3 All data are the average of duplicate shake flasks after 48 hours.Conditions: 40 ml medium/200 ml baffled shake flask, 4X Bioshieldcovers, 300 rpm, 2.5% inoculum and 43° C. Medium: 20 g/l Cargill 200/20soy flour, 1 × PSTE, 8 g/l (NH4)2SO4 and 5 g/l glutamate. Buffer: 0.1Mphosphate pH 7.2 and 0.3M MOPS pH 7.2. Carbon Source (Sterilizedseparately as 20 × stock): 60 g/l glucose or maltose w/10 mM Mg and 1.4mM Ca.

TABLE 9 10 liter fermentor evaluations of pantothenate productionstrains overexpressing serA or glyA. HMBPA (g/l) Pantothenate Added 3648 (g/l) run Strain Parent cassette hrs hrs 36 hrs 48 hrs P285 PA824 1825 41 46 P284 PA1012-4 PA824 serA 20 21 52 60 P286 PA1014-3 PA824 glyA14 16 71 86 P259 PA721B- 4 5 34 42 39 P287 PA1010-6 PA721B-39 serA 4 565 82 P289 PA1011-2 PA721B-39 glyA 2 3 56 79 P275 PA668-24 PA824 3 9 5572 The medium used is PFM-222. It is the same as medium PFM-155described in U.S. Ser. No. 60/262,995 (filed Jan. 19, 2001) except forthe following changes: (1) In the Batch Material: There is no Amberex1003. Cargill 200/20 (soy flour) 40 g/L has been changed to Cargill20–80 (soy grits) 50 g/L,MgSO₄.7H₂O is replaced with MgCl₂.7H₂0, 1 g/L,and SM-1000X is replaced with PSTE-1000X (PSTE-1000X = MnCl₂.4H₂O, 2.0g/L; ZnSO₄.7H₂O, 1.5 g/L; CoCl₂.6H₂O, 2.0 g/L; CuSO₄.5H₂O, 0.25 g/L;Na₂MoO₄.2H₂O, 0.75 g/L). In the Feed Material: SM-1000X is replaced withPSTE-1000X

Increasing pantothenate production can also be achieved by combiningoverexpression of serA and glyA in a single stain, and/or by introducinga mutation that leads to feedback resistant serA or glyA, or both.

Example VI Increasing the Expression of the GlyA Gene by Mutating thePurr Gene

As described in Examples III and V, expression of the glyA gene can beincreased by adding one or more copies of a cassette in which the glyAgene is driven by a strong, constitutive promoter. An alternative methodto increase glyA expression is to alter its regulation. Literaturedescribing a glyA::lacZ fusion suggests that the glyA promoter is ofmoderate strength under normal conditions (about 400 Miller Units), butthat this promoter is capable of being induced to relatively high levels(1,800 Miller units) if its negative regulator, the purR gene, isdeleted (Saxild et al. (2001) J. Bacteriol. 183:6175-6183). Therefore,experiments were preformed to determine if glyA expression, andconsequently pantothenate production, could be increased by deletingpurR from a pantothenate production strain.

The B. subtilis purR gene was amplified from PY79 chromosomal DNA byPCR, and the resulting fragment was cloned into PvuII cleavedpGEM-5-Zf(+) vector DNA to give plasmid pAN835F (SEQ ID NO:26, FIG. 8).This step eliminated the PvuII sites at both ends of the insert, leavinga unique PvuII site in the middle of the purR open reading frame. Next,a blunt PCR DNA fragment containing the Gram positive kanamycinresistance gene from pAN363F (SEQ ID NO:27) was ligated into this uniquePvuII site of pAN835F to give pAN838F (SEQ ID NO:28, FIG. 9).

pAN838F was then transformed into PY79, PA668-24, and PA824, selectingfor kanamycin resistance at 10 mg/l to give new sets of strains namedPA1059, PA1060, and PA1061, respectively. It was shown by PCR that allnew isolates contained the disrupted purR::kan allele that was expectedfrom a double crossover event. Several isolates of PA1060 and PA1061were tested for pantothenate production in test tube cultures grown inSVY glucose plus β-alanine (Table 10). The best isolates derived fromPA668-24, PA1060-2 and PA1060-4, gave an improvement from 3.0 g/lpantothenate to 5.3 to 5.1 g/l, respectively, which is an increase of75%. Likewise, the best isolates derived from PA824, PA1061-1 andPA1061-2 gave an increase from about 3.1 g/l to 5.4 g/l, also a 75%gain. These results suggest that the glyA gene is substantially inducedin these new strains by disruption of the purR gene. Alternatively, theimprovements in pantothenate production in PA1060 and PA1061 may be dueto more complex pleiotropic effects. In either case, deregulation of thepurR regulon has a positive effect on pantothenate production.

In other embodiments, the purR disruption can be installed in otherpantothenate production strains, for example those that have anintegrated P₂₆serA allele or more than one copy of the P₂₆panBCD operon.The purR gene can also be used as a site for addition of desiredexpression cassettes, such as P26panB. One can also use resistance tothe guanine analogs, such as 8-azaguanine, as a selection for a purRmutation.

TABLE 10 Production of pantothenate and fantothenate by derivatives ofPA824 and PA668-24 containing disrupted purR, in test tube culturesgrown in SVY glucose plus 5 g/l β-alanine. new [pan] Strain inoculum*parent feature OD₆₀₀ [fan] g/l g/l PA668-24 cam 5, tet PA824 — 9 b.d.3.0 7.5 ″ cam 5, tet ″ — 12 b.d. 3.0 7.5 PA1060-1 cam 5, tet PA668-24purR::kan 14 0.14 4.5 7.5 PA1060-2 cam 5, tet ″ ″ 12 b.d. 5.3 7.5PA1060-3 cam 5, tet ″ ″ 12 b.d. 4.5 7.5 PA1060-4 cam 5, tet ″ ″ 16 0.115.1 7.5 PA824 tet 30 PA377 — 9 0.25 3.2 ″ ″ ″ — 11 0.22 3.0 PA1061-1 tet15 PA824 purR::kan 13 0.45 5.4 PA1061-3 ″ ″ ″ 14 0.39 5.4 PA1061-4 ″ ″ ″11 0.40 4.7 b.d. = below detection *concentration of antibiotics in thepetri plate from which the inoculum colony was taken

Example VII Overexpression of the serA Gene from a Non-AmplifiableCassette

This Example describes another method to increase serine production, inwhich a two step procedure deposits a strong, constitutive promoter(P₂₆) in front of the chromosomal serA gene. Two plasmids wereconstructed, each containing about 700 base pairs of DNA sequence fromthe region immediately upstream of the native sera gene. The firstplasmid, pAN821, also contains the 3′ half of the sera coding region,and in between the two aforementioned sequences, a kanamycin resistancegene (SEQ ID NO:30, FIG. 10). When transformed into B. subtilis,selecting for kanamycin resistance, pAN821 will give a disruption of thesera gene, leading to serine auxotrophy. This creates a genetic sequencetermed the ΔserA::kan allele.

The second plasmid, designed to introduce the P₂₆ sera structure, wasconstructed by inserting the serA upstream sequence at the 5′ end of theP₂₆ promoter in pAN395. The resulting plasmid, pAN824, is shown in FIG.11 (SEQ ID NO:31). The plasmid pAN395 is similar to pAN393 described inExample IV. The open reading frame of the sera gene was synthesized byPCR using B. subtilis PY79 DNA as the template. The upstream primercontains an XbaI site and a moderately strong synthetic ribosome bindingsite, RBS2. The downstream primer contains a BamHI site. This serA openreading frame was used to replace the panBCD genes in the medium copyplasmid, pAN006, to give pAN395 (SEQ ID NO:29, FIG. 12). This plasmidcontains the sera gene expressed from the P₂₆ promoter and the RBS2ribosome binding site.

The ΔserA::kan allele from pAN821 was introduced into strain PA824 togive PA1026. As expected, PA1026 did not grow on minimal medium. In thesecond step, the P₂₆ serA cassette from plasmid pAN824 was introducedinto PA1026, selecting for serine prototrophy, to give strain PA1028.Several PA1028 isolates were confirmed to have the expected chromosomalstructure (P₂₆ serA) by diagnostic PCR. These isolates were then testedfor pantothenate production in test tube cultures grown for 48 hours inSVY plus 5 g/l β-alanine (Table 11). The PA1028 isolates (derived fromPA824) gave increases from 10% to 25% in pantothenate production. Asshown in Table 12, in shake flask experiments, PA824 produced about 7g/l pantothenate, whereas: PA 1028 produced 11 g/l.

Example VIII Construction of Pantothenate Producing Strains that ContainBoth an Integrated Non-Amplifiable P₂₆ serA Cassette and an AmplifiableP₂₆glyA Cassette

Since a non-amplifiable P₂₆ serA cassette integrated at serA led tohigher pantothenate synthesis (see, e.g., Table 12), and since achloramphenicol amplifiable P₂₆ glyA cassette at glyA led to much higherpantothenate synthesis (see, e.g., PA1014-3, Table 8), it was, proposedthat a combination of the two might be synergistic. Strain PA1028-4,which is the derivative of PA824 that contains the non-amplifiable P₂₆serA cassette integrated at serA, was transformed to chloramphenicolresistance at 5 mg/l using chromosomal DNA from PA1014-3, to give a setof strains named PA1038, which now contain the chloramphenicolamplifiable P₂₆glyA cassette. PA1038 isolates were tested forpantothenate production using standard test tube cultures grown in SVYplus β-alanine (Table 13). As expected, PA1038 showed a dramaticincrease in pantothenate production from about 4.2 g/l by PA824 to 6.6to 7.5 g/l by the PA1038 set. Isolates PA1038-3 and PA1038-12 werefurther tested in shake flasks as shown in Table 12. Both produced anaverage of 13.6 g/l pantothenate, as compared to the 7.4 g/lpantothenate produced by PA824.

TABLE 11 Production of pantothenate and fantothenate by derivatives ofPA824 that contain a single copy of P₂₆ serA at the serA locus, in 48hour test tube cultures grown in SVY plus 5 g/l β-alanine. Strain parentOD₆₀₀ [fan] g/l [pan] g/l PA824 17 0.44 4.0 PA824 15 0.45 4.0 PA1028-1PA824 13 0.46 4.4 PA1028-2 ″ 18 0.49 4.9 PA1O28-3 ″ 15 0.44 4.4 PA1028-4″ 13 0.43 4.5 PA1028-5 ″ 14 0.45 4.4 PA1028-6 ″ 11 0.43 4.8 PA1028-8 ″15 0.51 5.0 b.d. = below detection

TABLE 12 Shake flask evaluation of pantothenate production strainsoverexpressing serA and/or glyA. glyA serA Fantothenate PantothenateStrain Parent cassette cassette (g/l) (g/l) PA824 0.6 7.4 PA1014-3 PA824N × P₂₆glyA 0.7 12.0 PA1028-4 PA824 P₂₆serA @ serA 0.8 11.1 PA1038-3PA1028-4 N × P₂₆glyA P₂₆serA @ serA 0.5 13.6 PA1038-12 PA1028-4 N ×P₂₆glyA P₂₆serA @ serA 0.6 13.6 All data are the average of duplicateshake flasks after 48 hours. Conditions: 40 ml medium / 200 ml baffledshake flask, 4 × Bioshield covers, 300 rpm, 2.5% inoculum and 43° C.Inoculum: SVY base w/maltose 24 hours at 43° C. Medium: 20 g/l Cargill200/20 soy flour, 8 g/l (NH₄)₂SO₄, 5g/l glutamate and 1 × PSTE. Buffer:0.1 M phosphate pH 7.2 and 0.3 M MOPS pH 7.2. Carbon Source (Sterilizedseparately as 20 × stock): 30 g/l maltose, 5 mM MgCl₂ and 0.7 mM CaCl₂.

TABLE 13 Pantothenate production by PA1038, a derivative of PA824 thatcontains a non-amplifiable P₂₆ serA cassette at serA and an amplifiabieP₂₆ glyA cassette at glyA. Inoculum Strain Medium OD₆₀₀ [Fan] g/L [Pan]g/L PA824 tet 15 16 0.56 4.4 PA824 ″ 14 0.59 4.3 PA824 tet 30 12 0.574.3 PA824 ″ 14 0.58 4.2 PA1038-3 cam 5, tet 15 16 0.47 7.2 PA1038-4 ″ 140.49 7.0 PA1038-5 ″ 15 0.52 7.0 PA1038-6 ″ 15 0.51 7.2 PA1038-9 ″ 140.56 7.2 PA1038-11 ″ 13 0.49 6.6 PA1038-12 ″ 16 0.58 7.5 Test tubecultures were grown with SVY glucose plus 5 g/l β-alanine at 43° C. for48 hours.

Example IX Increasing the Production of MTF by Altering the GlycineCleavage Pathway

As demonstrated with the above examples, increasing MTF production inbacteria increases the production of pantothenate in stains that havebeen engineered to produce more pantothenate by manipulation of thepanBCD and/or panE genes. It has been demonstrated that pantothenateproduction can be increased by increasing the expression of the glyA orthe serA gene. Stronger promoters or ribosome binding sites can be usedto increase glyA or sera expression as demonstrated in Examples IIIthrough V and VII through VIII. Alternatively, the expression of theglyA gene can be deregulated in Bacillus by disrupting the purRrepressor gene as illustrated in Example VI.

Another method to increase MTF production is to enhance the expressionof enzymes of the glycine cleavage pathway. For example, enzymes encodedby the gcvT, gcvPA, gcvPB, gcvH, and pdhD genes catalyze the breakdownof glycine to MTF, CO₂, and NH₃. A strong, constitutive promoter, suchas the SP01 phage P₂₆ promoter described previously, can be cloned infront of the gcvT-gcvPA-gcvPB operon or in front of the gcvH or pdhDgene to enhance their expression. In addition to the above mentionedapproaches, additional glycine, which is inexpensive, can be added tothe medium to further enhance MTF production by any strain engineered asdescribed herein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A process for the enhanced production of pantothenate, comprisingculturing a recombinant microorganism selected from the group consistingof Bacillus, Corynebacterium, Lactobacillus, Streptomyces, Salmonella,Escherichia, Klebsiella, Serratia, Proteus, and Saccharomyces having (i)a deregulated pantothenate biosynthetic pathway, and (ii) a deregulatedmethylenetetrahydrofolate (MTF) biosynthetic pathway, under conditionssuch that pantothenate production is enhanced as compared to a wild-typemicroorganism, wherein the deregulated pantothenate biosynthetic pathwayis achieved by overexpressing at least one gene selected from the groupconsisting of panB, panC, panD, and panE1, and wherein the deregulatedMTF biosynthetic pathway is achieved by overexpressing at least one geneselected from the group consisting of gcv, serA, serC, serB, glyA, sul,fol, mtrA, pab, panB and purR derived from a microorganism of the genusBacillus, Corynebacterium, Lactobacillus, Lactococci, or Streptomyces.2. The process of claim 1, wherein said microorganism has at least twopantothenate biosynthetic enzymes deregulated.
 3. The process of claim1, wherein said microorganism has at least three pantothenatebiosynthetic enzymes deregulated.
 4. The process of claim 1, whereinsaid microorganism has at least four pantothenate biosynthetic enzymesderegulated.
 5. The process of claim 4, wherein said microorganism has aderegulated ketopantoate hydroxymethyltransferase, a deregualtedketopantoate reductase, a deregulated pantothenate synthetase and aderegulated aspartate-α-decarboxylase.
 6. The process of claim 1,wherein said microorganism further has a deregulated isoleucine-valine(ilv) biosynthetic pathway.
 7. The process of claim 6, wherein saidmicroorganism has at least two isoleucine-valine (ilv) biosyntheticenzymes deregulated.
 8. The process of claim 6, wherein saidmicroorganism has at least three isoleucine-valine (ilv) biosyntheticenzymes deregulated.
 9. The process of claim 8, wherein saidmicroorganism has a deregulated acetohydroxyacid acid synthetase, aderegulated acetohydroxyacid isomeroreductase, and a deregulateddibydroxyacid dehydratase.
 10. The process of claim 1, wherein themicroorganism has at least one MTF biosynthetic enzyme deregulated. 11.The process of claim 10, wherein the microorganism has a deregulatedglyA gene.
 12. The process of claim 10, wherein the microorganism has aderegulated serA gene.
 13. The process of claim 10, wherein themicroorganism has a deregulated glyA gene and a deregulated serA gene.14. The process of claim 11 or 13, wherein the microorganism has amutated, deleted or disrupted purR gene.
 15. The process of claim 1,wherein said microorganism is cultured under conditions of excessserine.
 16. The process of claim 1, wherein the microorganism is a Grampositive microorganism.
 17. The process of claim 1, wherein themicroorganism belongs to the genus Bacillus.
 18. The process of claim 1,wherein the microorganism is Bacillus subtilis.
 19. The process of claim1, wherein the microorganism overexpresses relative to a wild-type cellat least one methylenetetrahydrofolate (MTF) biosynthetic enzyme encodedby glyA, serA, serC, serB, gcvT, gcvPA, gcvPB, or gcvH.
 20. The processof claim 14, wherein the microorganism overexpresses the glyA gene as aresult of a mutation in the purR repressor gene.
 21. The process ofclaim 14, wherein the microorganism further has a deregulatedmethylenetetrahydrofolate (MTF) biosynthetic enzyme encoded by glyA,serA, serC, serB, gcvT, gcvPA, gcvPB, or gcvH.
 22. The process of claim14, wherein the microorganism further has a deregulated pantothenatebiosynthetic pathway.
 23. The process of claim 22, wherein saidmicroorganism has at least two pantothenate biosynthetic enzymesderegulated.
 24. The process of claim 22, wherein said microorganism hasat least three pantothenate biosynthetic enzymes deregulated.
 25. Theprocess of claim 22, wherein said microorganism has at least fourpantothenate biosynthetic enzymes deregulated.
 26. The process of claim25, wherein said microorganism has a deregulated ketopantoatehydroxymethyltransferase, a deregualted ketopantoate reductase, aderegulated pantothenate synthetase and a deregulatedaspartate-α-decarboxylase.
 27. The process of claim 14, wherein saidmicroorganism further has a deregulated isoleucine-valine (ilv)biosynthetic pathway.
 28. The process of claim 27, wherein saidmicroorganism has at least two isoleucine-valine (ilv) biosyntheticenzymes deregulated.
 29. The process of claim 27, wherein saidmicroorganism has at least three isoleucine-valine (ilv) biosyntheticenzymes deregulated.
 30. The process of claim 29, wherein saidmicroorganism has a deregulated acetohydroxyacid acid synthetase, aderegulated acetohydroxyacid isomeroreductase, and a deregulateddihydroxyacid dehydratase.
 31. The process of claim 14, wherein saidmicroorganism is cultured under conditions of excess serine.
 32. Theprocess of claim 14, wherein the microorganism is a Gram positivemicroorganism.
 33. The process of claim 14, wherein the microorganismbelongs to the genus Bacillus.
 34. The process of claim 14, wherein themicroorganism is Bacillus subtilis.